1. Evolutionary Biology
  2. Structural Biology and Molecular Biophysics
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Indirect sexual selection drives rapid sperm protein evolution in abalone

  1. Damien Beau Wilburn  Is a corresponding author
  2. Lisa M Tuttle
  3. Rachel E Klevit
  4. Willie J Swanson
  1. University of Washington, United States
Research Article
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Cite this article as: eLife 2019;8:e52628 doi: 10.7554/eLife.52628

Abstract

Sexual selection can explain the rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if not directly involved in fertilization. In the marine mollusk abalone, sperm secrete enormous quantities of two rapidly evolving proteins, lysin and sp18, that are stored at nearly molar concentrations. We demonstrate that this extraordinary packaging is achieved by associating into Fuzzy Interacting Transient Zwitterion (FITZ) complexes upon binding the intrinsically disordered FITZ Anionic Partner (FITZAP). FITZ complexes form at intracellular ionic strengths and, upon exocytosis into seawater, lysin and sp18 are dispersed to drive fertilization. NMR analyses revealed that lysin uses a common molecular interface to bind both FITZAP and its egg receptor VERL. As sexual selection alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution.

Introduction

Genes associated with fertilization are often the fastest evolving in any genome (Swanson and Vacquier, 2002), and in mammals, spermatozoa-specific genes show the greatest divergence between species (Torgerson et al., 2002). While cooperation may be expected over conflict, differences in male and female reproductive strategies result in sexual arms races which can cause the rapid evolution of exaggerated sexual characters that have been hypothesized since Darwin as a driver of speciation (Andersson, 1994). Sexual selection acting on gamete recognition proteins is postulated to create reproductive barriers and facilitate speciation (Arnold and Houck, 2016; Wilburn et al., 2017), but sexual selection theory has not previously explained why sperm proteins that do not directly interact with the egg evolve rapidly. Here, we demonstrate that sexual selection can propagate through protein interaction networks and potentially drive global evolution of the sperm proteome.

The marine mollusk abalone is a classic system to study molecular barriers to hybridization (Lewis et al., 1982) and is the source of the first discovered pair of interacting reproductive proteins: sperm lysin and the egg vitelline envelope receptor of lysin (VERL) (Swanson and Vacquier, 1997). Animal eggs are surrounded by an extracellular barrier called the vitelline envelope (VE) that restricts the entry of sperm (the mammalian VE is referred to as the zona pellucida, ZP). VERL is a major component of the abalone VE which lysin dissolves by binding to repetitive domains within VERL (Raj et al., 2017; Swanson and Vacquier, 1997). Over millions of years, changes in VERL have resulted in positive sexual selection on lysin and a coevolutionary chase to maintain binding affinity. Extant lysins dissolve conspecific VEs more efficiently than those of closely related taxa, providing one mechanism of species-specific fertilization and a barrier to hybridization (Swanson and Vacquier, 1997; Vacquier and Lee, 1993). As VE dissolution is mediated by non-enzymatic lysin-VERL binding, the process is concentration dependent and sperm express enormous quantities of lysin (Lewis et al., 1982). A single male abalone can contain >1 gram of lysin, reflecting >0.1% of its total body weight. Lysin is stored in a specialized secretory granule termed the acrosome. Based on electron microscopy (Haino-Fukushima and Usui, 1986; Lewis et al., 1980) we estimate that the acrosomal concentration of lysin is ~0.1–1.0 M, in stark contrast to saturation concentrations of ~0.001 M under in vitro conditions (Wilburn et al., 2018).

Lysin is not the only highly abundant, rapidly evolving protein in the abalone sperm acrosome. Another is sp18, a fusogenic paralog of lysin that likely mediates plasma membrane fusion between egg and sperm (Swanson and Vacquier, 1995). The receptor of sp18 is unknown, but given its interaction with the abalone egg, its accelerated evolution is likely due to sexual selection (Aagaard et al., 2010). Sp18 is nearly as abundant as lysin, so it must also be packaged at high concentrations, yet its fusogenic properties make it even less soluble than lysin (Kresge et al., 2001). Recently, a new family of small acrosomal proteins termed sperm protein 6 kDa (sp6) was discovered by shotgun transcriptomics and proteomics (Palmer et al., 2013). While also rapidly evolving and hypothesized to evolve via sexual selection, initial efforts to identify an egg binding partner for sp6 were unsuccessful (Palmer, 2013). However, while lysin and sp18 are highly positively charged proteins (+12 to +24), isoforms of sp6 are highly anionic (−6 to −16) and include an N-terminal poly-aspartate region of variable length (1–11 residues). Given this charge complementarity, we hypothesized that sp6 may facilitate packaging of lysin and sp18 inside the sperm acrosome. We demonstrate that the rapid evolution of sp6 is due to intra-sperm protein coevolution with lysin and sp18 to allow for their dense storage in the acrosome via novel Fuzzy Interacting Transient Zwitterion (FITZ) complexes. Heterodimers of lysin-sp6 or sp18-sp6 form through hydrophobic interactions, and these heterodimers polymerize into large particles (diameter >100 nm) through ionic interactions of the complementary positive and negative charges. Upon secretion of the acrosomal contents into highly ionic seawater, FITZ complexes are disrupted, and the dispersal of lysin and sp18 facilitating fertilization. In light of its newly identified function, we have named sp6 the FITZ Anionic Partner (FITZAP).

Results

Different species of abalone express different numbers of FITZAP isoforms named for the length of the N-terminal poly-aspartate region (Palmer, 2013). In red abalone (Haliotis rufescens), two isoforms (FITZAP-4D and FITZAP-8D) result from alternative splicing of different versions of exon 1 (signal peptide and the N-terminus with the poly-aspartate region) with a common exon 2 (C-terminus) (Figure 1—figure supplement 1). Each isoform was purified using strong anion exchange (SAX) chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), with mass spectrometry revealing that both isoforms were smaller than their cDNA open reading frame predicted (~3–4 kDa vs ~6 kDa). The observed masses are consistent with proteolytic processing of FITZAP by the Golgi enzymes furin and carboxypeptidase B (Figure 1—figure supplement 2). Despite their high net positive charges, both lysin and sp18 co-eluted with FITZAP at high-salt concentrations by SAX chromatography (Figure 1). Particularly striking is that sp18 (+22) eluted at higher salt concentrations than lysin (+12), which mirrors the elution profiles of FITZAP-4D (−10) and FITZAP-8D (−8), respectively, suggesting isoform-specific interactions. While purified lysin showed no affinity for anion exchange resin, its elution was retarded when mixed with either FITZAP-4D or FITZAP-8D in vitro, albeit less dramatically than the ex vivo samples (Figure 1—figure supplement 3). This is likely a consequence of sample preparation and the extraordinary concentration of lysin and FITZAP inside the acrosome compared to in vitro reconstitutions, allowing more complexes to persist during the chromatography. Together, these findings support that in vivo interactions likely enable co-purification of lysin/sp18 and FITZAP from sperm lysate.

Figure 1 with 3 supplements see all
Co-purification of FITZAP and cationic acrosomal proteins by anion exchange.

(A) SDS-PAGE of proteins from red abalone sperm lysate separated by anion exchange. Despite their high net positive charges, lysin and sp18 co-eluted with FITZAP isoforms in the highest salt fractions. (B) RP-HPLC analysis of select anion exchange fractions which contained lysin. The peak of lysin elution at 300 mM coincides with the peak elution of FITZAP-8D, with both sp18 and FITZAP-4D eluting under higher salt conditions.

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate how FITZAP interacts with the cationic fertilization proteins. We focused on lysin and FITZAP-8D because (A) they showed the strongest coelution by SAX chromatography (Figure 1), (B) lysin is more soluble than sp18 (Aagaard et al., 2010; Swanson and Vacquier, 1995), (C) interactions with its egg receptor have been characterized (Raj et al., 2017; Wilburn et al., 2018), and (D) a solution structure has been determined (Wilburn et al., 2018). Chemical shift analysis of FITZAP-8D revealed that it is an intrinsically disordered protein (IDP); even when bound to lysin, FITZAP-8D remained highly dynamic and adopted no regular secondary structure (Figure 2—figure supplements 1 and 2). Thus, lysin and FITZAP form a fuzzy complex: protein complexes that exist in an ensemble of different interchanging configurations (Figure 2). Formation of the fuzzy complex is primarily due to packing between hydrophobic amino acids in residues 21–29 of FITZAP (Figure 3A) and an exposed hydrophobic face on lysin near the nexus of the N- and C-termini. Covering this hydrophobic patch with FITZAP imparts a high net negative charge to this region of lysin, likely explaining the affinity of lysin to anion exchange resins when FITZAP is present. Significantly, the FITZAP-binding region of lysin is also the same surface that recognizes its egg receptor VERL, based on both chemical shift perturbations (Figure 2A) and paramagnetic relaxation enhancement (PRE) data (Figure 2—figure supplement 3). This exposed hydrophobic surface is likely why purified lysin has a much lower in vitro solubility limit compared to the acrosome where FITZAP is also highly abundant.

Figure 2 with 3 supplements see all
Lysin recognizes VERL and FITZAP through a common binding interface.

(A) NMR perturbation of red lysin upon conspecific binding of either VERL repeat 1 (data from Wilburn et al., 2018) or FITZAP-8D (1.5 molar equivalents). Two regions near residue 60 and residue 100 of lysin are perturbed in both cases (dashed line: median + 1.5 * interquartile range). (B) Mapping of perturbation onto a lysin solution structure (PDB 5utg) shows spatial clustering of these two regions to a single binding surface of lysin. (C) Docking of VERL repeat 1 (based on PDB 5mr3 by Raj et al., 2017) and FITZAP-8D (based on restraints from paramagnetic relaxation enhancement) supports that FITZAP is an intrinsically disordered protein that shields the VERL binding interface of lysin.

Figure 3 with 1 supplement see all
NMR perturbation of FITZAP-8D upon lysin binding.

(A) Backbone amide chemical shift perturbation of FITZAP-8D upon addition of lysin under seawater conditions (500 mM NaCl). The solid line denotes the relative hydrophobicity of the sequence which correlates well with the chemical shift perturbation, supporting that FITZAP-lysin interactions under high-salt conditions are driven by hydrophobic packing. The poly-aspartate region is highlighted in red and shows no chemical shift perturbation under seawater conditions. (B) Intensity perturbation of FITZAP-8D by binding of lysin under different ionic strengths ranging from approximately intracellular levels (150 mM) to seawater conditions (500 mM). Differences in intensity perturbation as a function of salt are in part driven by the salt dependence on lysin-FITZAP Kd (Figure 5), but the weak perturbation observed in the poly-aspartate region only under low salt conditions support that these residues are involved in some form of molecular interaction with lysin (likely salt bridges as part of FITZ complexes) only under intracellular conditions.

Given a single interaction surface for two different binding partners, lysin must separately recognize FITZAP inside the acrosome and, upon secretion, VERL at the VE. As abalone is a marine mollusk, we postulated that local inorganic salt concentrations may play an important role. The total salt concentration of seawater is ~500 mM, yet the intracellular environment of marine animals is less concentrated. Osmolality is maintained in marine vertebrates by active transport of water and ions, whereas many invertebrates such as abalone are osmoconformers and use high intracellular concentrations of free amino acids, betaines, and other highly soluble metabolites to achieve osmolality (but not isotonicity) (Venter et al., 2018). While the exact intracellular concentration of inorganic ions in abalone sperm is unknown, it is likely much lower than seawater based on data from sea urchin gametes (Horthschild and Barnes, 1953; Rodríguez and Darszon, 2003) and other abalone tissues (Jia and Liu, 2018). The inorganic salt concentration may be even lower in the acrosome where the acrosomal proteins – given their extraordinary concentrations and net charges – may themselves serve as osmolytes. To compare how lysin-FITZAP interactions are influenced by differing environmental contexts, we performed biophysical experiments under low (150 mM NaCl) and high (500 mM NaCl) salt concentrations that approximate the intracellular or seawater environments, respectively. Under both conditions, the hydrophobic patch of FITZAP interacts with lysin, yet NMR intensity perturbation of the poly-aspartate region was only observed under low-salt concentrations where intermolecular salt bridges may form (Figure 3B). Differences in NMR perturbation establish that lysin and FITZAP undergo slower subunit exchange under low-salt concentrations (Figure 3—figure supplement 1). Equimolar mixtures of lysin and FITZAP-8D under low-salt conditions form extremely large oligomers with an average diameter of ~400 nm (compared to a mean lysin diameter of ~6 nm); these large particles are not present at high-salt concentrations (Figure 4). Our data establish that lysin and FITZAP associate hydrophobically to form fast-exchanging, fuzzy heterodimers that are essentially zwitterionic. These heterodimers that we call Fuzzy Interacting Transient Zwitterions (FITZs) can form intermolecular salt bridges that allow tight packaging under intracellular-like conditions. Upon secretion into seawater, the FITZ complexes are disrupted and subunit exchange rate increases, allowing lysin to be rapidly liberated from FITZAP, permitting interactions with VERL via a common binding interface. The formation and dissolution of FITZ complexes based on the environmental context provides a mechanism for exceptionally high packaging concentrations of lysin inside the sperm acrosome as well as its rapid dispersal in seawater when fertilization may be imminent.

FITZ complex formation is dependent on both FITZAP and the ionic environment.

Dynamic light scattering measurements of lysin with and without equimolar FITZAP-8D at intracellular (150 mM) and seawater (500 mM) salt conditions. Lysin and FITZAP associate into FITZ complexes with mean diameter of ~400 nm only under intracellular salt conditions.

Lysin and VERL are rapidly coevolving sperm and egg proteins that exhibit species-specific interactions (Wilburn and Swanson, 2016). Given the common binding interface, we postulated that there may be similar coevolution between lysin and FITZAP. While such coevolution was not detected using sequence-based approaches (Figure 5—figure supplement 1), the small size and intrinsic disorder of FITZAP can reduce statistical power of such analyses. A functional consequence of lysin and FITZAP coevolution would be species-specific interactions where the proteins from the same species have higher binding affinities compared to heterospecific pairs, as observed between lysin and VERL. Using fluorescence polarization, binding affinities were measured between lysin and all FITZAP isoforms for three abalone species (red, disk, and green). Like red abalone, green abalone has two FITZAP isoforms but with shorter poly-aspartate regions (1D and 4D), while disk abalone has a single FITZAP isoform with an even longer poly-aspartate array (11D). Lysin had greater affinity for the high-D FITZAP isoforms compared to low-D forms. For all three species, lysin bound the conspecific high-D isoforms of FITZAP with equilibrium dissociation constants of ~1–2 μM under intracellular salt conditions. Except for disk lysin and red FITZAP-8D, all cases of heterospecific binding were significantly weaker, supporting lysin-FITZAP coevolution (Figure 5A). Both hydrophobic and ionic interactions likely contribute to these higher affinity complexes, yet the specificity of disk FITZAP-11D (with the longest poly-aspartate array) to disk lysin supports that electrostatic attraction alone is not sufficient to explain FITZ complex formation. Under seawater conditions, there was a ≥ 20 fold decrease in conspecific binding affinity (Figure 5B), consistent with the liberation of lysin from FITZ complexes after its release from the acrosome.

Figure 5 with 1 supplement see all
FITZAP interactions with cationic acrosomal proteins within and between species.

(A) Lysin-FITZAP interactions were measured by fluorescence polarization under approximately intracellular salt conditions for all combinations within and between species of red, disk, and green abalone (conspecific interactions are shaded as red, blue, and green, respectively). Low micromolar binding affinities were observed for conspecific interactions of lysin with high-D FITZAP isoforms. (B) Binding affinities between conspecific lysin and FITZAP high-D isoforms are substantially reduced under extracellular seawater conditions. (C) Green sp18 shows tighter binding to conspecific FITZAP-4D compared to FITZAP-1D; however, the relative affinity of sp18 for 4D over 1D (77/22 = 3.5 X) is greater than that of lysin (>100 X), suggesting that sp18 has higher preference for low D isoforms compared to lysin. (n.s. = not significant at p<0.05; n.d. = not determined if anisotropy was not monotonically positive and consistent with single-state binding; Kd reported as mean ± standard error).

As sp18 likely facilitates the fusion of egg and sperm plasma membranes (Swanson and Vacquier, 1995), it is more hydrophobic than lysin and may therefore be even more reliant on a partner for storage and dispersal. Lysin and sp18 are paralogs with similar tertiary structures that have subfunctionalized following gene duplication (Kresge et al., 2001; Swanson and Vacquier, 1995), so the multiple FITZAP isoforms may also have subfunctionalized to bind these different paralogs. As lysin showed greater affinity for high-D FITZAP isoforms (Figure 5A), we hypothesized that sp18 may be coevolving with low-D isoforms. We indeed observe correlated rates of molecular evolution between sp18 and low-D FITZAP isoforms, consistent with coevolution (Figure 5—figure supplement 1). While the highly fusogenic sp18 is mostly insoluble when purified (Aagaard et al., 2010; Swanson and Vacquier, 1995), we were able to measure conspecific affinities between sp18 and FITZAP isoforms from green abalone. While green sp18 also bound green FITZAP-4D more tightly than FITZAP-1D (Figure 5C), its relative affinity for 4D over 1D (~3.5X) is substantially less compared to the relative affinity of 4D to 1D for lysin (>100X). For our anion exchange experiments in red abalone, we observed tighter co-elution between lysin/FITZAP-8D and sp18/FITZAP-4D (Figure 1). Therefore, both evolutionary and biochemical evidence support that FITZAP isoforms have subfunctionalized to respond to the divergent evolutionary trajectories of lysin and sp18.

Discussion

We propose a system of tethered coevolution between VERL, lysin, and FITZAP where VERL imposes direct sexual selection on lysin and indirect sexual selection on FITZAP (summarized in Figure 6). As with any coevolving system, there is likely reciprocity with all binding partners imposing some form of selection on one another; however, we choose to focus on the unidirectional case of VERL influencing lysin influencing FITZAP for several reasons. First, sexual selection theory has emphasized ‘female choice,’ because the higher energetic cost of oocytes in most species favor greater mate selectivity (Arnold and Houck, 2016). These assumptions are most apt when discussing genes involved in fertilization. Second, lysin evolves ~5X faster than its coevolving regions of VERL (Galindo et al., 2003), suggesting that it is experiencing greater directional selection than VERL. Lysin, but not VERL, is also monomorphic within some abalone populations and experienced recent selective sweeps (Clark et al., 2009). Third, in sea urchins (another organism with broadcast spawning), longitudinal measurements of allele frequencies for gamete recognition proteins support that female proteins will shift to lower affinity interactions in response to increased polyspermy risk, and high-affinity binding is restored by adaptation of male proteins (Levitan et al., 2019). This suggests that evolutionary dynamics of lysin are more responsive to VERL than vice versa. Fourth, as FITZAP is an IDP whose function is to facilitate the storage and dispersal of fertilization proteins, we anticipate reduced conservation on its primary sequence from intramolecular epistasis, with its evolution mostly being driven by directional selection imposed by its binding partners.

Rapid evolution of FITZAP is due to indirect sexual selection from VERL.

Male abalone have overlapping habitat ranges and spawning periods such that there is opportunity for hybridization to occur. Sperm are attracted to eggs via chemotaxis and bind to the vitelline envelope (A). At this stage, the acrosome is still intact (Lewis et al., 1982) with lysin and FITZAP tightly packaged as FITZ complexes. Binding to the VE causes the sperm to acrosome react (B), releasing its contents into highly ionic seawater which disperses lysin and FITZAP. Liberated lysin can now dissolve the VE by binding VERL domains (C); these interactions are species-specific and provide one barrier to hybridization (Swanson and Vacquier, 1997). Because the rapid evolution of lysin is driven by direct sexual selection to maintain sperm-egg interactions, and FITZAP is co-evolving to bind the same interface, VERL imposes indirect sexual selection onto FITZAP. Microscopy images adapted from Lewis et al. (1982) and Sakai et al., 1982.

Because IDPs lack a single favored conformation, they experience less purifying selection (or slower evolution relative to genetic drift) to maintain a tertiary fold and have lower sequence conservation (Brown et al., 2011). Beyond relaxed purifying selection and greater rates of genetic drift, IDPs also experience more positive selection compared to structural domains (Afanasyeva et al., 2018), presumably in response to coevolution with their binding partners. We expect FITZAP to respond more to selection from lysin than vice versa. It has been suggested that intrinsic disorder may be a mechanism of adaptation to shifts in environmental conditions; for example, host-changing parasites have higher genome-wide levels of predicted protein disorder compared to obligate intracellular parasites and endosymbiotes (Pancsa and Tompa, 2012). The change in ionic strength experienced by secreted proteins of most marine animals is likely an extreme example of such shifts between chemical environments. The intrinsic disorder of FITZAP may be crucial to its apparent structural versatility and high evolvability in response to a rapidly coevolving partner.

Across animals, plants, and microbes, genes associated with fertilization usually evolve faster than the rest of the genome (Swanson and Vacquier, 2002; Wilburn et al., 2017; Wilburn and Swanson, 2016). Like macroscopic secondary sex characters, direct sexual selection can drive elaboration of molecular phenotypes such as the extraordinary abundance of lysin and sp18. Combined with sequence differences that yield species-specific interactions with coevolving receptors, these proteins are one of many reproductive barriers that likely contribute to speciation. Given the necessity of fertilization for sexually reproducing taxa, few selective pressures are likely stronger than sexual selection, and even its indirect effects through coevolutionary networks are likely substantial. In this report, we demonstrated how indirect sexual selection drives rapid evolution of a sperm protein not associated with fertilization. Indirect selection may further propagate throughout the sperm proteome and be a general mechanism to explain accelerated gametic protein evolution.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Escherichia coli)5-alphaNew England BiolabsC2987Chemically competent cells for cloning and sequencing
Strain, strain background (Escherichia coli)Rosetta2Novagen71400Chemically competent cells for protein expression
Biological sample (Haliotis rufescens)red abalone spermThe Abalone FarmFreshly isolated from male abalone testis
Biological sample (Haliotis fulgens)green abalone lysinCollected from 32°51’00’ N, 117°16’34’ WPurified from sperm by cation exchange chromatography, and provided by Vic Vacquier
Recombinant DNA reagentpCR4-TOPOThermo-FisherK457540Cloning of DNA for sequencing
Recombinant DNA reagentpET11dNovagen69439Cloning of DNA for protein expression
Recombinant DNA reagentMBP-FITZAPThis paperSee Supplementary file 1
Sequence-based reagentFITZAP F’This paper5’-ATGAGGGTTRTTCTAATT-3’
Sequence-based reagentSp18 F’This paper5’-GGAAACAGTATGAGGTYTTTGSTGCTT-3’
Peptide, recombinant proteinMBP-TEV proteaseSigma-AldrichT4455
Commercial assay or kitBCA Protein AssayPierce23225
Software, algorithmRStudio v.1.0.136https://rstudio.com/RRID:SCR_000432
Software, algorithmRAxML v8.2.12https://cme.h-its.org/exelixis/software.htmlRRID:SCR_006086
Software, algorithmFast Statistical Alignment (FSA) v.1.15.9http://fsa.sourceforge.net/RRID:SCR_016114
Software, algorithmPAML v4http://abacus.gene.ucl.ac.uk/software/paml.htmlRRID:SCR_014932
Software, algorithmNMRFAM-Sparkyhttps://nmrfam.wisc.edu/nmrfam-sparky-distribution/
Software, algorithmXplor-NIH v.2.48https://nmr.cit.nih.gov/xplor-nih/
Software, algorithmTALOS-Nhttps://spin.niddk.nih.gov/bax/software/TALOS-N/
Software, algorithmPyMOL v.1.8https://github.com/schrodinger/pymol-open-sourceRRID:SCR_000305

Purification and mass spectral characterization of natural FITZAP

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Natural FITZAP was purified and characterized based on methods modified from Palmer et al. (2013). Briefly, sperm were collected by dissection of testes from red abalone (Haliotis rufescens) and lysed by trituration in 1% Triton X-100 (w/v)/250 mM NaCl/2 mM EDTA/10 mM MES, pH 6, and centrifuged at 3200 x g, 8°C for 30 min. The supernatant was applied to a 10 mL CM52 cellulose column (Whatman, Maidstone, UK) to remove lysin and other positively charged proteins. The FITZAP-enriched flow through was diluted with 2 volumes of 50 mM Tris, pH 8, applied to a 10 mL Q sepharose column (Sigma-Aldrich, St. Louis, MO), and protein fractions collected by gravity flow with a stepwise NaCl gradient buffered with 20 mM Tris, pH 8. Fractions were analyzed by 15% Tris-Tricine SDS-PAGE (Schägger and von Jagow, 1987), and FITZAP localized to fractions with ≥300 mM NaCl. These fractions were pooled and concentrated using 3 kDa centrifugal ultrafilters (EMD-Millipore, Billerica, MA), and individual components purified by reverse phase high performance liquid chromatography (RP-HPLC) using a Vydac C18 column (0.46 × 15 cm; Hichrom, Berkshire, UK) that was eluted from 0 to 70% acetonitrile in 0.1% trifluoracetic acid at 1% acetonitrile per minute. Individually purified proteins were analyzed by LC/MS-MS using data-dependent acquisition on an LTQ Velos tandem mass spectrometer (Thermo Scientific, Waltham, MA) for determination of intact protein and fragment ion masses.

Sequence analysis of FITZAP and estimation of molecular evolutionary rates

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Draft genome assemblies are available for disk abalone (Haliotis discus) (Nam et al., 2017) and red abalone (H. rufescens) (Masonbrink et al., 2019). The contigs or scaffolds containing FITZAP exons were identified by performing BLAST searches (Camacho et al., 2009) using FITZAP open reading frames as queries. These genomic regions were extracted with an additional 5 kb of flanking sequence on both the 5’ and 3’ ends, and aligned using fsa v1.15.9 (Bradley et al., 2009). For molecular evolutionary analysis, available cDNA sequences for lysin, VERL, sp18, and FITZAP were downloaded from Genbank (Accession # M34388, M59969-M59972, M98875, AF453553, AF490761-AF490763, AF490765, AF490766, L36552, L36554, L36589, KC752594, KC752595, KC752597-KC752602). We additionally sequenced sp18 cDNA by RT-PCR from black abalone (Haliotis cracherodii), flat abalone (Haliotis wallallensis), and disk abalone (H. discus), and FITZAP from flat abalone. Abalone testis cDNA was provided by Jan Aagaard, and RT-PCR was performed using an oligo-dT reverse primer using a sp18 specific (5’-GGAAACAGTATGAGGTYTTTGSTGCTT-3’) or FITZAP-specific (5’-ATGAGGGTTRTTCTAATT-3’) forward primer. PCR products were cloned into a pCR4-TOPO vector (Invitrogen), transformed into 5-alpha competent E. coli (New England Biolabs, Ipswich, MA), and plasmid DNA from at least four clones of each transformation were supplied to Eurofins Scientific (Louisville, KY) for Sanger sequencing. No sequence variation was observed between clones, and these sequences have been deposited into Genbank (Accession # MN102340-MN102343). A maximum likelihood gene tree was constructed with RAxML v8.2.12 (Stamatakis, 2014) using the PROTGAMMALG substitution model and a concatenation of protein sequences from lysin, VERL, and sp18 from six abalone species (red, flat, disk, black, pinto (Haliotis kamtschatkana), and green (Haliotis fulgens)) aligned by Clustal Omega (Sievers et al., 2011), and used as a representative of the likely species tree. Based on methods by Clark et al. (2009), support for co-evolution between protein coding genes was evaluated by weighted linear regression using branch dN/dS values estimated with PAML v4 (Yang, 2007) for each of the four genes (lysin, VERL, sp18, and FITZAP), with FITZAP further divided into low-D and high-D isoforms.

Cloning and expression of recombinant FITZAP

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Recombinant FITZAP was expressed in E. coli and purified to near homogeneity using multiple chromatography steps. Given the low molecular weight of different FITZAP isoforms, expression in E. coli required fusion to a larger carrier protein from which FITZAP could be removed by enzymatic proteolysis and purified. FITZAP isoforms from red, disk, and green abalone were genetically fused by PCR with a maltose binding protein (MBP) cassette containing an N-terminal 6xHis tag and a C-terminal linker sequence followed by a tobacco etch virus (TEV) protease cleavage site (see Supplementary file 1 for sequences). Recombinant FITZAP proteins included an Amino Terminal Cu- and Ni-binding tag (ATCUN) to facilitate protein purification, improve TEV proteolysis (Kapust et al., 2002), and permit collection of NMR PRE constraints (Donaldson et al., 2001). The combined MBP-FITZAP construct was cloned into the pET11d expression vector (Novagen, San Diego, CA), transformed into Rosetta2 chemically competent E. coli (EMD-Millipore, Billerica, MA) which express additional tRNA genes for Lys and Arg that are abundant in abalone genes, and clones validated by Sanger sequencing (Eurofin Genomics, Louisville, KY). For expression of unlabeled FITZAP, E. coli clones were cultured in LB media supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37°C, 250 rpm; when cultures reached an optical density at 600 nm (OD600) of ~0.6, recombinant protein expression was induced by addition of IPTG to a final concentration of ~100 µM, cells harvested by centrifugation after 3.5 hr, and stored at −20°C. For expression of isotopically labeled FITZAP, methods were adapted from Wilburn et al. (2018). Briefly, E. coli clones expressing MBP-FITZAP were cultured in LB media supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37°C, 250 rpm until the OD600 reached ~0.4; cells were then collected by centrifugation, concentrated 4-fold into M9 media with 20 µM FeSO4 and 100 µg/mL ampicillin without carbon or nitrogen sources, and maintained at 37°C, 250 rpm for 35 min to deplete the cells of free unlabeled amino acids; cultures were then supplemented with 3 g/L ammonium sulfate (14N or 98% 15N) and 4 g/L glucose (12C or 99% 13C) for 35 min to regenerate amino acid stores with appropriate isotopes; then expression was induced by addition of IPTG to a final concentration of ~100 µM, cells harvested by centrifugation after 3.5 hr, and stored at −20°C. Cell pellets were then lysed by sonication in 1% octylthioglucoside/50 mM NaCl/50 mM Tris, pH 8, then supplemented with 0.2 mg/mL lysozyme for 30 min, centrifuged @ 3.2 k x g for 2 hr, the supernatant clarified by passage through a 0.2 µm PES filter, and the filtrate applied to a 10 mL Ni-NTA column (Pierce, Rockford, IL) equilibrated in 500 mM NaCl/20 mM Tris/1 mM imidazole, pH 8. The column was subsequently washed with increasing concentrations of imidazole in 500 mM NaCl/20 mM Tris, pH 8: six column volumes (CVs) at 1 mM imidazole, 2 CVs at 20 mM, 1 CV at 40 mM, 1 CV at 60 mM, and MBP-FITZAP eluted using 3 CVs at 200 mM imidazole. The elution fraction was buffer exchanged using a YM30 centrifugal ultrafilter (Millipore, Billerica, MA) into 100 mM NaCl/20 mM Tris, pH 8, supplemented with TEV Protease (Sigma-Aldrich) at an enzyme:substrate ratio of ~1:500 by mass, and incubated overnight at room temperature with gentle mixing. Following proteolysis, precipitate was removed by centrifugation at 3.2 k x g for 30 min, and the supernatant applied to a 10 mL Ni-NTA column equilibrated in 500 mM NaCl/20 mM Tris/1 mM imidazole, pH 8. The column was washed with 3 CVs of 500 mM NaCl/20 mM Tris/1 mM imidazole, pH 8, a FITZAP-enriched fraction eluted using 3 CVs of 500 mM NaCl/20 mM Tris/20 mM imidazole, pH 8, and MBP/MBP-FITZAP enriched fraction eluted using 500 mM NaCl/200 mM Tris/1 mM imidazole, pH 8. The 20 mM imidazole FITZAP-enriched fraction was further purified by size-exclusion chromatography (G-75 superfine; Pharmacia, Piscataway, NJ) followed by strong anion exchange chromatography (Mono-Q; Pharmacia) on an Agilent 1100 HPLC with UV detection at 220 nm.

Recombinant lysin expression

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Recombinant lysin from red and disk abalone was expressed and purified by methods from Wilburn et al. (2018). Briefly, lysin coding sequences were cloned into the pET11d expression vector (Novagen), transformed into Rosetta2 chemically competent E. coli (EMD-Millipore, Billerica, MA) which express additional tRNA genes for Lys and Arg that are essential for lysin expression, and clones validated by Sanger sequencing (Eurofin Genomics, Louisville, KY). To provide flexibility in isotopic labeling for NMR experiments, lysin expression was performed in cultures where (1) biomass with high ribosome densities was produced by initially culturing in complex media, (2) the cells were concentrated ~4X in minimal media without nitrogen or carbon to deplete amino acid stores, (3) ammonium sulfate (14N or 15N) and glucose (uniformly 12C or 13C) were added to regenerate amino acids with the appropriate isotopes, then (4) expression was induced by addition of IPTG. Growth under minimal media conditions provides complete removal of the N-terminal methionine from endogenous E. coli methionine aminopeptidase activity, leaving a single exogenous Gly on the N-terminus that has no detectable impact on lysin structure or function. Properly folded recombinant lysin was expressed into inclusion bodies that were isolated by centrifugation of cell lysate, washed to remove contaminant proteins, denatured in 5M guanidinium hydrochloride, refolded by rapid dilution, and purified using cation exchange chromatography.

Ion exchange purification of lysin and sp18

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Methods for lysin purification were adapted from Lewis et al. (1982). For both natural and recombinant lysin, step chromatography was performed using CM52 cellulose (Whatman) equilibrated in 250 mM NaCl/10 mM MES/2 mM EDTA, pH 6. For recombinant lysin, methods are described above for refolding from inclusion bodies. For natural lysin, H. rufescens sperm were isolated by dissection of male testes and lysed by trituration in 0.1% Triton X-100/250 mM NaCl/10 mM MES/2 mM EDTA, pH 6; insoluble material (including chromatin) was removed by centrifugation at 3200 x g for 30 min. Crude fractions of natural or recombinant lysin were applied to a CM52 cellulose column, rinsed with >6 CVs of 250 mM NaCl/10 mM MES/2 mM EDTA, pH 6, and eluted with 3 CVs of 1 M NaCl/10 mM MES/2 mM EDTA, pH 6. Purified natural lysin and sp18 from H. fulgens was generously supplied by Vic Vacquier. Purified lysin and sp18 was concentrated and buffer exchanged to 150 mM NaCl/10 mM Tris, pH 7.4 using YM10 centrifugal ultrafilters (Millipore).

Comparison of conspecific/heterospecific FITZAP-Lysin/Sp18 interactions by fluorescence polarization

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Purified recombinant FITZAP proteins from red, disk, and green abalone were buffer exchanged into 0.5 mL phosphate buffered saline (PBS) using a 3 kDa centrifugal ultrafilter (Millipore), and fluorescently labeled by addition of 18 μL Alexa Fluor 488 SDP (Invitrogen, Carlsbad, CA) at 10 mg/mL DMSO at 4°C overnight with mixing. Fluorescently labeled FITZAP was separated from free fluorophore by size exclusion using Nap5 columns (GE Life Sciences, Piscataway, NJ). Protein concentrations for labeled FITZAP and unlabeled lysin were determined by BCA Protein Assay (Pierce). Each fluorescently labeled FITZAP isoform was standardized to 1 µM, lysin added to concentrations of 0, 1, 2, 5, 10, 15, 20, 30, 40, and 50 µM, and fluorescence anisotropy measured using a Fluorolog spectrofluorometer (Horiba Scientific, North Edison, NJ). All species/isoform combinations between FITZAP and lysin were performed in 150 mM NaCl/10 mM Tris, pH 7.4, and when possible, anisotropy measurements were collected in technical duplicate (although sample degradation over the course of the experiment prevented this for all combinations). For conspecific pairings with low µM binding affinities, anisotropy measurements were repeated in 500 mM NaCl/10 mM Tris, pH 7.4. Additionally, anisotropy experiments were repeated for green FITZAP isoforms with green sp18 using the same series of concentrations as lysin in 150 mM NaCl/20 mM Tris, pH 7.4. Dissociation constants (Kd) were estimated for all combinations by nonlinear regression using the equation Anisotropy  ΔAmax[Lysin]+[FITZAP]+Kd([Lysin]+[FITZAP]+Kd)24[Lysin][FITZAP]2+Aintercept with the R function nlsLM in the package minpack.lm.

Anion exchange analysis of positively charged acrosomal proteins

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Preliminary experiments separating H. rufescens sperm lysate by anion exchange chromatography yielded the surprising result of both lysin and sp18 (highly positively charged proteins) adhering to the column and eluting at relatively high ionic strengths, and it was hypothesized that this unexpected observation may result from FITZAP supplying negative charges to these proteins as part of FITZ complexes at low ionic strength. Sperm from H. rufescens was isolated by dissection of testes, lysed by trituration in 2 mL of 0.1% Triton X-100/20 mM Tris, pH 8 supplemented with TURBO DNase (Ambion), and centrifuged at 2 k x g for 10 min. Clarified lysate was applied to a 5 mL Q Sepharose (Sigma-Aldrich) column, and 2 CV fractions collected at 0, 25, 50, 100, 200, 300, 400, and 600 mM NaCl in 20 mM Tris, pH 8. The same step gradient was performed after applying 2 mL aliquots of (1) red lysin at 0.5 mg/mL, (2) red lysin at 0.5 mg/mL with equimolar red FITZAP-8D, and (3) red lysin at 0.5 mg/mL with equimolar red FITZAP-4D. Anionic exchange fractions were separated by 15% Tris-Tricine SDS-PAGE (Schägger and von Jagow, 1987) and stained with Coomassie Brilliant Blue R-250.

NMR analysis of FITZAP-Lysin interactions

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Purified, isotopically labeled H. rufescens FITZAP-8D (15N/13C) was concentrated to ~0.2–1.0 mM in 50 mM NaCl/10 mM Tris, pH 7.4/7% D2O using a 3 kDa centrifugal ultrafilter (Millipore). For FITZAP, all NMR experiments were performed on a Bruker Avance 800-Mhz spectrometer fitted with a TCI CryoProbe (Bruker), while NMR experiments with labeled lysin were performed on a Bruker Avance 500-Mhz spectrometer (Bruker). NMR assignments of red FITZAP-8D were obtained using a combination of 2D/3D experiments: 15N- and 13C-filtered HSQC, HNCACB, CBCAcoNH, HNCO, HNHA, 15N-HSQC-TOCSY, and 15N-HSQC-NOESY. Spectra were processed using NMRpipe (Delaglio et al., 1995) and analyzed using NMRFAM-SPARKY (Lee et al., 2015). Assignments were 70% complete for backbone atoms (91% excluding the poly-aspartate region). Chemical shift indices were calculated using TALOS-N (Shen and Bax, 2013). To characterize lysin binding residues, 15N- and 13C-HSQC spectra of 15N/13C-FITZAP-8D (200 μM in 500 mM NaCl/10 mM Tris, pH 7.4/7% D2O) were acquired at six concentrations of recombinant monomeric lysin (Wilburn et al., 2018) from 0 to 500 μM. Chemical shift perturbations (CSPs) between 15N-HSQC spectra were calculated as (Δ1H)2+(0.1Δ15N)2. The interaction of lysin and salt with FITZAP was assessed by acquiring 15N- and 13C-HSQC spectra of 15N/13C-FITZAP-8D (200 μM in 10 mM Tris, pH 7.4/7% D2O) with or without monomeric lysin (140 µM) at different salt concentrations (150–500 mM in 50 mM steps). Reciprocal titration experiments were performed with 15N- and 13C-HSQC spectra acquired for 15N/13C-monomeric lysin (150–200 μM in 10 mM Tris, pH 7.4/7% D2O) at different salt concentrations (150 or 500 mM) with varying concentrations of FITZAP-8D (0 to 600 µM). To obtain intermolecular PRE constraints from the FITZAP-8D N-terminal ATCUN motif, R2 relaxation rates were measured for 15N- monomeric lysin (150 µM in 500 mM NaCl/10 mM Tris, pH 7.4/7% D2O) with equimolar FITZAP-8D with or without 135 µM CuSO4 using delays of 8.48, 16.96, 25.44, 33.92, 42.40, 50.88, and 59.36 ms. Data has been deposited in the BMRB (27962).

Structural analysis

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Using Xplor-NIH 2.48 (Schwieters et al., 2006; Schwieters et al., 2003), a structural ensemble of lysin and FITZAP-8D heterodimers was modeled by simulated annealing from 4000 to 25 K with torsion dynamics followed by Cartesian minimization using constraints from PRE measurements and degenerate CSP pairings (adapted from Clore and Schwieters, 2003). For comparison, a similar ensemble was constructed between lysin and VERL repeat 1 using a lysin solution structure (PDB 5utg) and a VERL repeat 1 crystal structure (PDB 5ii4) using constraints based on a cocrystal structure of lysin and VERL repeat 3 (PDB 5mr3). Figures of 3D protein models were produced using PyMOL (v.1.8, Schrodinger, LLC), regular secondary structure defined using the DSS function, and electrostatic surfaces calculated using the APBS/PDB2PQR server (Dolinsky et al., 2004; Jurrus et al., 2018).

Characterization of FITZ complexes by dynamic light scattering

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Dynamic light scattering measurements were performed using a Zetasizer Nano (Malvern Pananalytical). Natural lysin purified from red abalone testis lysate was standardized to 100 µM in 10 mM Tris, pH 7.4, and light scattering was measured with and without equimolar FITZAP-8D at different salt concentrations (150 and 500 mM NaCl). Six technical replicates of 15 scans each were collected and averaged. FITZAP-8D in isolation produced no substantial light scattering over buffer.

References

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    Sexual Selection
    1. M Andersson
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    Princeton: Princeton University Press.
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    The inorganic constituents of the sea-urchin egg
    1. L Horthschild
    2. H Barnes
    (1953)
    Journal of Experimental Biology 30:534–544.
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    The Sperm Proteome of the Red Abalone, Haliotis Rufescens, and the Discovery and Characterization of an Abundant, Rapidly Evolving Acrosome Protein
    1. MR Palmer
    (2013)
    Seattle: University of Washington.
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Decision letter

  1. Patricia J Wittkopp
    Senior Editor; University of Michigan, United States
  2. Hannes Neuweiler
    Reviewing Editor; University of Würzburg, Germany
  3. Hannes Neuweiler
    Reviewer; University of Würzburg, Germany
  4. Kristaps Jaudzems
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper reports an unusual function of an intrinsically disordered domain in facilitating storage of fertilization proteins in abalone sperm and in their release upon exposure to sea water. The authors combine NMR spectroscopy with fluorescence polarization and dynamic light scattering to show that the highly charged and intrinsically disordered domain organizes the storage of a fertilization protein at extremely high concentration in sperm, which is required for egg penetration. The malleable and disordered domain condenses fertilization proteins in soluble form by targeting the same interaction interface as the egg protein and by compensating repulsive electrostatic forces in oligomeric assemblies through provision of opposite charges. Upon release into sea water, high concentrations of salt screen charge interactions thus facilitating disassembly and fertilization. Besides uncovering an intriguing mechanism of soluble protein condensation, the authors show that rapid co-evolution can proceed indirectly through protein-protein interaction networks. The study is of broad interest because it integrates topics of intrinsic protein disorder, evolutionary biology, protein biochemistry and biophysics.

Decision letter after peer review:

Thank you for submitting your article "Indirect sexual selection drives rapid sperm protein evolution in abalone" for consideration by eLife. Your article has been reviewed by two peer reviewers, including Hannes Neuweiler as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Patricia Wittkopp as the Senior Editor. The following individual involved in review of your submission has also agreed to reveal their identity: Kristaps Jaudzems (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Review synthesis:

The manuscript "Indirect sexual selection drives rapid sperm protein evolution in abalone" by Wilburn et al. reports on the discovery and characterization of the functional role of the small, intrinsically disordered domain sp6 in storage and release of the abalone sperm protein lysin, which participates in fertilization. Wilburn et al. use NMR spectroscopy, fluorescence polarization and dynamic light scattering to investigate structure and interaction of sp6 bound to lysin in-vitro and its response to changes in salt concentration. The dependence of complex formation on salt concentration explains liberation of lysin upon exocytosis into seawater. The authors propose a mechanism whereby the negatively charged sp6 binds to the positively charged lysin and generates a zwitter-ionic complex that allows storage of lysin at extraordinary high concentrations. Solubility is facilitated by formation of large, multimeric assemblies that are held together by attractive charge-charge interactions. Given the novel functional role the authors rename sp6 as fuzzy interacting transient zwitterion anionic partner (FITZAP). Using NMR spectroscopy they find that the binding interface of lysin is common for both, FITZAP and the egg vitelline envelope receptor of lysin (VERL). Determination of binding affinities between FITZAP and lysin from three different species probed by fluorescence polarization indicates similar species-specificity as lysin-VERL interactions. Based on their findings the authors propose an indirect sexual selection mechanism resulting in co-evolution of FITZAP to maintain binding of lysin, which co-evolves with VERL via direct sexual selection. The mechanism is proposed to generally explain rapid sperm protein evolution.

Reproductive proteins are encoded by fast evolving genes. Yet, rapid coevolution of fertilization proteins that are not directly involved in interactions with the egg is puzzling. Moreover, it is a mystery how sperm can store lysin at internal concentrations approaching 1 M required for successful fertilization. The study by Wilburn et al. provides new interesting details about the binding mode and interaction specificities of the FITZAP proteins allowing the authors to elucidate function and solubility within the sperm acrosome, and to explain their rapid evolution even though they are not directly involved in fertilization. The study is carefully designed and experiments are thoroughly performed. Furthermore, the manuscript is well written and results are properly interpreted.

However, the following issues need to be addressed before publication:

1) The authors claim to demonstrate how the extraordinary packaging of lysin and sp18 within the acrosome is achieved. However, while the salt conditions used for NMR studies correspond to intracellular levels, the protein concentrations were much lower than intracellular. Therefore, the obtained results may not fully depict the protein complexes that are formed within the acrosome. Is it possible to concentrate the complex to at least 10 mM concentration and record NMR data to see, which residues lose intensity?

2) DLS measurements indicate formation of oligomers with mean diameter of 400 nm, which does not fit with the heterodimeric model presented in Figure 1C. Can the NMR data be interpreted in a way to describe the oligomer structure that should have more than one interaction interface?

3) It would be interesting to see what the 400 nm diameter oligomers look like under an electron microscope, especially because the size distribution is rather homogeneous.

4) At the beginning of the Results section the authors show that FITZAP has a molecular weight of 3-4 kDa (35 residues, Figure 2—figure supplement 1) and argue for it being an intrinsically disordered domain. But given the small size of FITZAP one may argue that it is actually a peptide rather than a domain. Most peptides are unfolded. Is it really appropriate to assign FITZAP to the class of intrinsically disordered domains?

5) In the Results section, paragraph two, the authors argue that incubation of lysin with FITZAP would lead to binding of the complex to an anion exchange column and retard lysin. But in Figure 1—figure supplement 3 there is hardly any retardation evident. Samples from higher ionic strength eluates show hardly protein bands in SDS-PAGE (hardly visible in the gel images) and, if at all, traces of a complex. This appears reasonable because the total number of negative charges of FITZAP does not fully compensate the higher number of positive charges of lysin.

6) Figure 2—figure supplement 1: The authors argue that FITZAP is disordered when free and bound to lysin. But what are the concentrations probed in NMR data shown in this figure? This should be stated in the figure caption. Are the authors sure that they saturated binding of FITZAP to lysin by applying sufficient excess concentration of lysin?

7) Figure 1C, lower panel: in the structural model the dimensions of FITZAP-8D and lysin appear similar. This is confusing because FITZAP is a 35-residue peptide (or domain) while lysin is a 125-residue domain.

8) In paragraph three of the Results section (Figure 3—figure supplement 1) the authors infer kinetics of FITZAP-lysin association/dissociation from NMR chemical shift perturbation. But this is not valid. No accurate kinetic data can be inferred from the data shown. In order to measure kinetics the authors would need to carry out rapid-mixing experiments (possibly requiring a stopped-flow machine) using e.g. fluorescence polarization as a probe for association/dissociation. Rate constants of dissociation could be measured using chasing experiments in combination with rapid-mixing. Anyway, in my opinion, no support from kinetic data is required for the conclusions of this work. It is reasonable to assume that the rate constant of dissociation of the complex, once it is released from sperm to sea water environment, is substantially faster than diffusion and binding of lysin to the egg cell receptor VERL.

9) In the Results paragraph ten the authors state that lysin has a higher affinity for high-D FITZAP isoforms compared to low-D isoforms. This finding suggests that not only hydrophobics but also the charged tail of FITZAP plays a role in binding to lysin. Lysin and FITZAP are net oppositely charged and will attract each other in solution, which will lead to electrostatically assisted binding (see e.g. Schreiber and Fersht, Nat Str Biol 1996, 3, 427-431; Janin, Proteins 1997, 28, 153-161; Schreiber et al., Chem Rev 2009, 109, 839-860).

10) In paragraph one of the Discussion the authors state that they anticipate minimal evolutionary selection on the primary sequence of FITZAP because it is a disordered domain with low sequence variation. Does this not contradict their main conclusion saying that indirect sexual selection acts on FITZAP?

11) A general point: the density of data shown in main figures is rather low. The manuscript was probably originally written up concisely for a journal with very limited space. The authors could move supplemental figures into the main manuscript, which would be beneficial for the reader and improve the overall quality.

https://doi.org/10.7554/eLife.52628.sa1

Author response

However, the following issues need to be addressed before publication:

1) The authors claim to demonstrate how the extraordinary packaging of lysin and sp18 within the acrosome is achieved. However, while the salt conditions used for NMR studies correspond to intracellular levels, the protein concentrations were much lower than intracellular. Therefore, the obtained results may not fully depict the protein complexes that are formed within the acrosome. Is it possible to concentrate the complex to at least 10 mM concentration and record NMR data to see, which residues lose intensity?

We have been unable to concentrate lysin beyond ~1mM. Experiments conducted using low molecular weight cutoff centrifugal ultrafilters yielded the same final concentration of lysin regardless of salt concentration and/or presence of equimolar FITZAP. This may be due to fuzzy condensates being disrupted upon centrifugation and/or sufficient exchange of FITZ complexes still permitting lysin aggregation. However, these concentrations were not required to detect both chemical shift and intensity changes by 15N-HSQC (more intensity loss in low salt, more CSPs in high salt, due to differences in exchange rate, see Figure 3—figure supplement 1).

2) DLS measurements indicate formation of oligomers with mean diameter of 400 nm, which does not fit with the heterodimeric model presented in Figure 1C. Can the NMR data be interpreted in a way to describe the oligomer structure that should have more than one interaction interface?

The model displayed in Figure 1C (now Figure 2C) reflects the dimeric state of lysin-FITZAP based on CSPs measured in high salt. The available NMR data can partially describe the larger oligomeric condition. In a titration series of labeled FITZAP with unlabeled lysin at different salt concentrations, we only observe intensity loss in the poly-aspartate region under low salt conditions, while there is consistent intensity loss (and CSPs) in the more hydrophobic C-terminal region of FITZAP regardless of salt concentration. Combined with the DLS data, this supports that hydrophobic interactions facilitate the initial heterodimer formation while ionic interactions enable the formation of the larger oligomeric species. These data are summarized in Figure 3. Reciprocal experiments with labeled lysin and unlabeled FITZAP at different salt conditions primarily report on the initial heterodimeric condition. While we see the same lysin residues being perturbed in both low and high salt, the formation of high molecular weight FITZ complexes results in line broadening beyond detection, which is supported by the ~10% median intensity loss in 150 vs 500 mM NaCl (Figure 3—figure supplement 1A).

3) It would be interesting to see what the 400 nm diameter oligomers look like under an electron microscope, especially because the size distribution is rather homogeneous.

We agree this would be interesting, but is likely not possible due to the dynamic nature of FITZ complexes. As our NMR experiments support that the FITZ complexes are transient and rapidly exchanging, it would be difficult to find equilibrium conditions where they would stably adhere to an EM grid. Furthermore, the fuzzy nature of lysin-FITZAP interactions likely would preclude any form of particle selection and averaging to gain additional structural insights.

4) At the beginning of the Results section the authors show that FITZAP has a molecular weight of 3-4 kDa (35 residues, Figure 2—figure supplement 1) and argue for it being an intrinsically disordered domain. But given the small size of FITZAP one may argue that it is actually a peptide rather than a domain. Most peptides are unfolded. Is it really appropriate to assign FITZAP to the class of intrinsically disordered domains?

Given its length, a case could be made for designating FITZAP as a peptide instead of a protein, yet in either scenario it is still an IDP (intrinsically disordered protein/peptide). As domains are defined based on a folded 3D structure, IDPs by definition lack domains and we do not use that term in the manuscript. While FITZAP likely falls in the grey area between proteins and peptides, we favor the use of “protein” as it is a naturally synthesized polypeptide chain with a now defined function.

5) In the Results section, paragraph two, the authors argue that incubation of lysin with FITZAP would lead to binding of the complex to an anion exchange column and retard lysin. But in Figure 1—figure supplement 3 there is hardly any retardation evident. Samples from higher ionic strength eluates show hardly protein bands in SDS-PAGE (hardly visible in the gel images) and, if at all, traces of a complex. This appears reasonable because the total number of negative charges of FITZAP does not fully compensate the higher number of positive charges of lysin.

This is an excellent point and further explanation has been added to the Results section. The difference between the ex vivo and in vitro results likely has to do with sample preparation, initial protein concentration, and the FITZ complex dissociation rate. Through several experiments to optimize the results, we found that both retention of lysin and sp18 on the anion exchange column was partially dependent on (1) length of centrifugation prior to applying the sample to the column (where FITZ complexes were presumably large enough to sediment), and (2) time to complete the chromatography (where less lysin/sp18 would be retained the longer they were on-column). To maximize the observed effect, sperm lysates were prepared by trituration of sperm in a small volume of Tris-buffered detergent solutions lacking NaCl to minimize ionic disruption of complexes, addition of DNaseI to reduce the sample viscosity, and a very brief centrifugation step to remove only the largest insoluble particles. By lysing in a near-minimal volume in the absence of salt, FITZ complexes should both be near their maximum concentration and experience slower dissociation. In contrast, our in vitro reconstitutions are limited by the solubility of lysin, such that we never achieve the extreme conditions of the naturally purified samples. The detection of any retardation under conditions orders of magnitude below the physiological concentrations supports that our ex vivo results are biologically meaningful.

6) Figure 2—figure supplement 1: The authors argue that FITZAP is disordered when free and bound to lysin. But what are the concentrations probed in NMR data shown in this figure? This should be stated in the figure caption. Are the authors sure that they saturated binding of FITZAP to lysin by applying sufficient excess concentration of lysin?

Figure 2—figure supplement 1 is not at saturation conditions, as it is not possible to saturate FITZAP and collect triple resonance spectra due to both lysin solubility limits and line broadening of FITZAP backbone amides at the binding interface. However, we have now added an additional supplemental figure (Figure 2—figure supplement 2) of FITZAP 13C-HSQC with different concentrations of lysin, including one near saturation (92%). Notably few 13C chemical shifts are observed in the CA-HA region, with the largest being A27, M34, and F35 before they broaden/overlap beyond detection. In both the CB-HB and methyl regions, CSPs are only observed in the 1H dimension, corroborating that FITZAP does not adopt secondary structure in the presence of lysin. Furthermore, based on our model of the lysin-FITZAP fuzzy heterodimer, these perturbed residues are localized near several aromatic residues on lysin (including H61, W62, Y65, and W68) such that changes in CA/HA chemical shifts may be due to ring current effects and broadening from chemical exchange.

7) Figure 1C, lower panel: in the structural model the dimensions of FITZAP-8D and lysin appear similar. This is confusing because FITZAP is a 35-residue peptide (or domain) while lysin is a 125-residue domain.

This is a consequence of the more linear FITZAP being compared to the more globular lysin.

8) In paragraph three of the Results section (Figure 3—figure supplement 1) the authors infer kinetics of FITZAP-lysin association/dissociation from NMR chemical shift perturbation. But this is not valid. No accurate kinetic data can be inferred from the data shown. In order to measure kinetics the authors would need to carry out rapid-mixing experiments (possibly requiring a stopped-flow machine) using e.g. fluorescence polarization as a probe for association/dissociation. Rate constants of dissociation could be measured using chasing experiments in combination with rapid-mixing. Anyway, in my opinion, no support from kinetic data is required for the conclusions of this work. It is reasonable to assume that the rate constant of dissociation of the complex, once it is released from sperm to sea water environment, is substantially faster than diffusion and binding of lysin to the egg cell receptor VERL.

The chemical shift is itself a rate that must be interpreted relative to the chemical exchange rate between free and bound states, hence differences in observed NMR peak centers and linewidths in fast (Δω << kex), intermediate (Δω ~ kex), and slow (Δω >> kex) exchange regimes. Comparing changes in observed chemical shift and peak intensities/linewidths is informative of the relative exchange regime (see Dwek, 1973). Changes in observed chemical shift are more pronounced under high salt compared to greater intensity loss under low salt, suggesting a change in kex (assuming similar Δω’s under both conditions, since presumably the lysin backbone amides are experiencing the same altered chemical environment upon hydrophobic packing). We make no specific references to the absolute magnitude of this change, only their relative qualitative difference.

9) In the Results paragraph ten the authors state that lysin has a higher affinity for high-D FITZAP isoforms compared to low-D isoforms. This finding suggests that not only hydrophobics but also the charged tail of FITZAP plays a role in binding to lysin. Lysin and FITZAP are net oppositely charged and will attract each other in solution, which will lead to electrostatically assisted binding (see e.g. Schreiber and Fersht, Nat Str Biol 1996, 3, 427-431; Janin, Proteins 1997, 28, 153-161; Schreiber et al., Chem Rev 2009, 109, 839-860).

We agree that both hydrophobic and ionic interactions facilitate lysin-FITZAP interactions to associate into high molecular weight FITZ complexes, and this is discussed in paragraph three of the Results section. The text has also been expanded in paragraph four. The length of the poly-D array alone does not predict lysin-FITZAP association by FP (e.g. Red Lysin binds Red FITZAP-8D efficiently, but not Disk FITZAP-11D; similarly, Green Lysin binds Green FITZAP-4D but not Red FITZAP-4D). This is consistent with a two-step binding event where sequence-specific hydrophobic interactions result in lysin-FITZAP heterodimers that then electrostatically associate into higher order FITZ complexes.

10) In paragraph one of the Discussion the authors state that they anticipate minimal evolutionary selection on the primary sequence of FITZAP because it is a disordered domain with low sequence variation. Does this not contradict their main conclusion saying that indirect sexual selection acts on FITZAP?

We have better clarified this point in the text. As an IDP, FITZAP likely lacks strong intramolecular epistatic purifying selection which most protein-coding genes experience to maintain a tertiary fold (and hence its function); instead, most of its evolution is likely due to intermolecular coevolution with binding partners.

11) A general point: the density of data shown in main figures is rather low. The manuscript was probably originally written up concisely for a journal with very limited space. The authors could move supplemental figures into the main manuscript, which would be beneficial for the reader and improve the overall quality.

Supplemental figures 3A and 5 have now been moved to the main text (now Figure 1 and 3).

https://doi.org/10.7554/eLife.52628.sa2

Article and author information

Author details

  1. Damien Beau Wilburn

    Department of Genome Sciences, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing
    For correspondence
    dwilburn@u.washington.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1255-9982
  2. Lisa M Tuttle

    Department of Biochemistry, University of Washington, Seattle, United States
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8889-232X
  3. Rachel E Klevit

    Department of Biochemistry, University of Washington, Seattle, United States
    Contribution
    Resources, Supervision, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3476-969X
  4. Willie J Swanson

    Department of Genome Sciences, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared

Funding

Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01-HD076862)

  • Willie J Swanson

Eunice Kennedy Shriver National Institute of Child Health and Human Development (K99-HD090201)

  • Damien Beau Wilburn

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Jan Aagaard, Emily Killingbeck, Jolie Carlisle, Alberto Rivera, Richard Feldhoff, Auberon Lopez, and Harmit Malik for feedback on the research and/or manuscript. We would also like to thank Vic Vacquier for providing purified acrosomal proteins from green abalone. This work was supported by National Institutes of Health grants R01-HD076862 to WJS and K99-HD090201 to DBW.

Senior Editor

  1. Patricia J Wittkopp, University of Michigan, United States

Reviewing Editor

  1. Hannes Neuweiler, University of Würzburg, Germany

Reviewers

  1. Hannes Neuweiler, University of Würzburg, Germany
  2. Kristaps Jaudzems

Publication history

  1. Received: October 10, 2019
  2. Accepted: December 20, 2019
  3. Accepted Manuscript published: December 23, 2019 (version 1)
  4. Version of Record published: January 9, 2020 (version 2)

Copyright

© 2019, Wilburn et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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