Structure-function relationship of trypanosomatid PYKs.

(A.) Schematic representation of a trypanosome, with a focus on their glycosome biochemistry. PYK catalyzes the last reaction of the trypanosomal glycolysis and is located outside of the glycosomes. (B.) The PYK monomer with the different domains color-coded and the domain boundaries shown. The pivot point for the AC core rotation (residues 430-433) is indicated by a magenta arrow. The substrate and effector binding sites are highlighted by yellow and cyan boxes, respectively. (C.) Schematic representation of the “rock and lock” model. The different PYK domains are color-coded as in panel (B.). In the absence of substrates (PEP and ADP) and effectors (F26BP or F16BP), trypanosomatid PYKs reside in a T-state (red box). The binding of substrates causes the enzyme to “rock” (R-state boxed in yellow). This consists of several structural rearrangements across the entire PYK tetramer that involve i) AC-core rotation of 6°-8° (with residues 430-434 as a pivot point), ii) closing of the lid domain (rotation of 30°-40°), iii) stabilisation of the AA’ dimer interfaces, and iv) flipping of the Arg311 side chain as part of remodeling the catalytic pocket for substrate accommodation. The binding of effectors to PYK’s C domain generates a “lock” in addition to the “rock”. This prompts the enzyme to adopt a conformation primed for eZcient catalysis (R-state boxed in blue); this involves i) the 6°-8° AC-core rotation, ii) stabilisation of the CC’ dimer interfaces, and iii) the Arg311 flip. The presence of substrates and effectors “rock and lock” the enzyme in the R-state (green box). The ribbon representations in the inset are the tetramer structures of T and R state PYK, superposed on the four pivot points. The AA’ and CC’ dimer interfaces are indicated by dashed lines. All structures and schematics were based on the crystal structures of apo and holo TcoPYK (this work and Pinto Torres et al., 2020).

sdAb42 selectively inhibits TcoPYK.

(A.) Effect of the addition of various concentrations of sdAb42 (red bars), sdAb44 (green bars), or sdAb BCII10 (grey bars) on the activity of TcoPYK prior to addition of substrates and effectors. The results demonstrate that only sdAb42 abrogates TcoPYK activity. (B.) Effect of the addition of sdAb42 (red bars) or sdAb44 (green bars) at a 1,000-fold molar excess on the activities of the various human PYK isoforms (M1PYK, human skeletal muscle isoform 1; M2PYK, human skeletal muscle isoform 2; LPYK, human liver; RPYK, human red blood cell). No impact of either sdAbs on enzyme activity could be observed.

sdAb42 binds and stabilizes the TcoPYK T state.

(A.) Cartoon representation of the sdAb42-TcoPYK complex observed in the crystal, in which one TcoPYK tetramer is bound by four copies of sdAb42. The TcoPYK domains are color-coded as in Figure 1 and sdAb42 is depicted in red. (B.) Close-up of the interaction between a single sdAb42 copy (cartoon representation) and AA’ dimer interface TcoPYK subunits (surface representation). sdAb42 and TcoPYK are color-coded as in panel (A.). The sdAb42 CDR1, CDR2, and CDR3 are colored in blue, green, and orange, respectively. (C.) Stereo view of the signature interactions made by Arg311 at the AA’ interface for TcoPYK in its T (TcoPYK-sdAb42, colored as in panels (A.) and (B.)) and R state (TcoPYK-citrate, colored in light grey; PDB ID 6SU1 (Pinto Torres et al., 2020)). Residues Arg263, Gly264, Gln298, Arg311 and Asp316 are shown in stick representation. The residues originating from the A’ domain are indicated by an asterisk ‘*’. (D.) Detailed view of the sdAb42 epitope in T and R state TcoPYK. sdAb42 is shown in surface representation and color-coded as in panel (A.). The residues constituting the sdAb42 epitope are shown in stick representation and colored in light grey (R state TcoPYK) or color-coded as in panels (A.) and (B.) (T state TcoPYK). A residue-by-residue comparison reveals that the epitope is significantly distorted in R state TcoPYK. (E.) CD spectra of apo TcoPYK (left panel, grey traces) and the sdAb42:TcoPYK complex (right panel, red traces) collected at different temperatures. The black dotted arrow represents the effect of the increasing temperature on the mean residue ellipticity measured at 222 nm, plotted in the inset (filled circles and dashed line represent the experimental data points and fit, respectively).

Perturbation analysis reveals distinct allosteric communication pathways in T and R state TcoPYK.

(A., C.) Surface representation of the TcoPYK tetramer in its T (A.) and R state (C.). The residues are color-coded according to their allosteric coupling intensity (ACI) values. The sdAb42 and effector molecule binding sites are delineated in red and cyan, respectively. (B., D.) The left panel depicts a cartoon representation of the TcoPYK tetramer in its T (B.) and R state (D.) colored in light grey. The residues constituting the active site and effector binding site are shown in sphere representation and colored in yellow and cyan, respectively. The residues that form the top 3 allosteric communication paths (top right) are also shown in sphere representations and colored in orange, green, and dark red (the dark red and green paths overlap, which is why the dark red paths are not visible). The bottom right panel shows a schematic depiction of the inter- (B.) and intrasubunit (D.) allosteric communication pathways. The AA’ dimer interface subunits are colored in dark and light grey, respectively, the active and effector binding sites are indicated by the yellow and cyan spheres, respectively, and the communication pathways are represented by the magenta arrows.

The sdAb42 epitope is conserved in trypanosomatid PYKs.

(A.) Surface representation of the TcoPYK AA’ dimer interface monomers. The residues are color-coded according to their CONSURF conservation score based on a multiple sequence alignment of trypanosomatid PYKs (Supplementary Figure 2). The sdAb42 epitope is delineated in red. (B.) ITC measurements at 25°C for the binding of sdAb42 to TcoPYK (left panel), LmePYK (middle panel) and TbrPYK (right panel). The top panels represent the thermograms in which the black lines depict the raw data. The bottom panels show the isotherms. The black dots display the experimental data points, and the red traces show the fit. (C.) Effect of the addition of increasing concentrations of sdAb42 on the activity of TcoPYK (red bars), LmePYK (pink bars) and TbrPYK (grey bars) prior to addition of substrates and effectors. The results demonstrate that sdAb42 abrogates the activities of all tested trypanosomatid PYKs. The inset displays the effect of sdAb42 on TcoPYK activity at lower sdAb concentrations.

Data collection and refinement statistics.

Statistics for the highest resolution shell are shown in parentheses.

Results of the in silico LlLlG analysis.

The LlLlG analysis was performed by uploading the sdAb42:TcoPYK structure to the mCSM-PPI2 (Rodrigues et al., 2019), mCSM-AB2 (Myung et al., 2020b), and mmCSM-AB (Myung et al., 2020a) servers and implementing the mutations of interest as specified by the author’s instructions (http://biosig.lab.uq.edu.au/tools). Calculations were performed for those epitope residues that differ between TcoPYK, LmePYK, and TbrPYK. The single Ile352Val and triple Lys43Gln/Val348Ala/Ile352Leu mutants correspond to changes the TbrPYK and LmePYK epitopes, respectively.

Thermodynamic parameters determined via analysis of the ITC data.

All titrations were performed in triplicate at 25°C (298.15 K).

List of interactions between sdAb42 and TcoPYK.

The # symbol indicates the number of times the interaction was observed over the total of six sdAb42:TcoPYK complexes present in the asymmetric unit. The average distances are only given for hydrogen bonds or electrostatic interactions.

Primer sequences employed for the RT-PCR experiments.

Slow binding inhibition kinetics.

Full kinetic time traces for the reaction catalyzed by TcoPYK,LmePYK, and TbrPYK (red, pink, and grey traces, respectively) in the presence of fixed substrate/effector concentrations and increasing sdAb42 concentrations. Only a subset of the traces is shown for the sake of clarity. The following curves are shown (from bottom to top): TcoPYK (0.15 nM sdAb42, 500 nM sdAb42, 750 nM sdAb42, 1000 nM sdAb42, 1500 nM sdAb42, 2000 nM sdAb42, no enzyme control), LmePYK (5 nM sdAb42, 750 nM sdAb42, 1250 nM sdAb42, 1500 nM sdAb42, 2500 nM sdAb42, 3000 nM sdAb42, no enzyme control), and TbrPYK (1 nM sdAb42, 1000 nM sdAb42, 1750 nM sdAb42, 2000 nM sdAb42, 3500 nM sdAb42, 4000 nM sdAb42, no enzyme control). The top inset shows a zoomed view of the activity curves to highlight the biphasic features of the traces. The bottom inset shows the IC50 determination by only taking into account the rates at longer time ranges. The three independent inhibition assay replicates for each enzyme are indicated by the filled triangles, squares, and circles, respectively.

The intracellular production of sdAb42 generates a growth defect in T. brucei.

(A.) The top panel schematically depicts the principle underlying the tetracycline (Tet) controlled production of the sdAb-mCherry fusion protein. The panels in the bottom left show fluorescence microscopy pictures of transgenic trypanosomes prior to (“no Tet”) and after Tet addition (“Tet-induced”) for an sdAb42 “high expressor” clone, an sdAb42 “low expressor” clone, and an sdAb BCII10 “high expressor” clone. The panels in the bottom right show growth curves recorded for these clones under culture conditions without (“no Tet”) and with Tet (“Tet-induced”). (B.) Median fluorescence intensity (MFI) values for all obtained transgenic sdAb42 (55 clones) and sdAb BCII10 (42 clones) monoclonal parasite lines. Growth curves were measured for four selected sdAb42 and sdAb BCII10 clones (indicated by the pink spheres; sdAb42: clones 1, 28,54, and 55; sdAb BCII10: clones 15, 16, 38, and 42). (C.) Results for the growth curves recorded for the clones highlighted in panel (B.) under culture conditions without (“no Tet”) and with Tet (“Tet-induced”). The clones were ranked from left to right based on the MFI values, which acts as a proxy for in situ intrabody levels (depicted by the gradient-colored triangle below the growth curves).

Comparison of the effector binding sites of different TcoPYK structures.

First panel from the top: Effector binding site of TcoPYK bound to fructose 2,6-bisphosphate (FBP; PDB ID: 6SU2). Second panel from the top: effector binding site of TcoPYK bound to sdAb42 (no sulfate, this work, PDB ID: 8RTL). Third panel from the top: effector binding site of TcoPYK bound to sdAb42 and sulfate prior to refinement (this work, PDB ID: 8RVR). Fourth panel from the top: effector binding site of TcoPYK bound to sdAb42 and sulfate after refinement (this work, PDB ID: 8RVR). In panels 2 to 4, the green and purple/blue density represent the (Fobs - Fcalc) and (2 Fobs - Fcalc) maps contoured at 3.10 (5 and 1.56 (5, respectively.

Amino acid sequence identities of trypanosomatid PYKs.

The amino acid sequence identities (expressed in percentage identity) resulting from a multiple sequence alignment are displayed under the form of a heat map.

Exponential decrease of sdAb42 protein levels over time.

(A.) Median fluorescence intensity (MFI) values for transgenic sdAb42-mCherry and sdAb BCII10-mCherry monoclonal parasite lines as a function of time. For sdAb42, a clear decreasing trend is observable, whereas sdAb BCII10 levels remain constant. (B) Western blot analysis of trypanosome cell lysates following a 5-day culture with (+) or without (-) 0.5 µg ml-1 tetracycline. The cell lysates were prepared from monoclonal parasite lines expressing the intrabodies. sdAb expression was revealed using an anti-HA HRP conjugated antibody, illustrating variable expression levels, i.e., a sdAb42 “high expressor” and “low expressor” clone whereas sdAb BCII10 is expressed at a stable high level. EF1-a was revealed as a reference control using a mouse anti-EF1-a antibody and an HRP-conjugated anti-mouse detection antibody.