1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

The inner mechanics of Rhodopsin Guanylyl Cyclase during 2cGMP-formation revealed by real-time FTIR spectroscopy

  1. Paul Fischer  Is a corresponding author
  2. Shatanik Mukherjee
  3. Enrico Peter
  4. Matthias Broser
  5. Franz Bartl
  6. Peter Hegemann
  1. Humboldt-Universität zu Berlin, Germany
  2. Center of Advanced European Studies and Research, Germany
  3. Humboldt University of Berlin, Germany
https://doi.org/10.7554/eLife.71384
Share this article
Cite this article
  1. Paul Fischer
  2. Shatanik Mukherjee
  3. Enrico Peter
  4. Matthias Broser
  5. Franz Bartl
  6. Peter Hegemann
(2021)
The inner mechanics of Rhodopsin Guanylyl Cyclase during 2cGMP-formation revealed by real-time FTIR spectroscopy
eLife 10:e71384.
https://doi.org/10.7554/eLife.71384
  2. Download BibTeX
  3. Download .RIS

Abstract

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

Data availability

Data files have been provided on Dryad (doi:10.5061/dryad.6wwpzgmzx) for Figure 4 and 5 as well as for the homology structures of the protein presented in the appendices, upon which figure 6 is based. This includes the protein prediction by AlphaFold 2.

The following data sets were generated

Article and author information

Author details

  1. Paul Fischer

    Humboldt-Universität zu Berlin, Berlin, Germany
    For correspondence
    paul.fischer.2@hu-berlin.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3766-9085

  2. Shatanik Mukherjee

    Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7359-9339

  3. Enrico Peter

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7913-5597

  4. Matthias Broser

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.

  5. Franz Bartl

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.

  6. Peter Hegemann

    Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3589-6452

Funding

Deutsche Forschungsgemeinschaft (221545957 B2+B5)

  • Franz Bartl
  • Peter Hegemann

Deutsche Forschungsgemeinschaft (SFB1315-327654276)

  • Peter Hegemann

Deutsche Forschungsgemeinschaft (390540038)

  • Peter Hegemann

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

Reviewing Editor

  1. Baron Chanda, Washington University in St. Louis, United States

Version history

  1. Received: June 17, 2021
  2. Accepted: October 18, 2021
  3. Accepted Manuscript published: October 19, 2021 (version 1)
  4. Accepted Manuscript updated: October 20, 2021 (version 2)
  5. Accepted Manuscript updated: October 25, 2021 (version 3)
  6. Version of Record published: November 8, 2021 (version 4)

Copyright

© 2021, Fischer et al.

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

Metrics

  • 1,149
    Page views
  • 217
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Paul Fischer
  2. Shatanik Mukherjee
  3. Enrico Peter
  4. Matthias Broser
  5. Franz Bartl
  6. Peter Hegemann
(2021)
The inner mechanics of Rhodopsin Guanylyl Cyclase during 2cGMP-formation revealed by real-time FTIR spectroscopy
eLife 10:e71384.
https://doi.org/10.7554/eLife.71384

Share this article

https://doi.org/10.7554/eLife.71384

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology
    2. Plant Biology

    Guanidine production by plant homoarginine-6-hydroxylases

    Dietmar Funck, Malte Sinn ... Jörg S Hartig
    Research Article

    Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

    1. Biochemistry and Chemical Biology
    2. Medicine

    Bone canonical Wnt signaling is downregulated in type 2 diabetes and associates with higher advanced glycation end-products (AGEs) content and reduced bone strength

    Giulia Leanza, Francesca Cannata ... Nicola Napoli
    Research Article

    Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156–0.366]) vs non-diabetic subjects 0.352% [0.269–0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46–30.10] vs non-diabetic subjects 76.24 MPa [26.81–132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=–0.7500, p=0.0255; r=–0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s modulus was negatively correlated with SOST (r=−0.5675, p=0.0011), AXIN2 (r=−0.5523, p=0.0042), and SFRP5 (r=−0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.

    1. Biochemistry and Chemical Biology

    The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

    Valentin Bohl, Nele Merret Hollmann ... Axel Mogk
    Research Article

    Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during, e.g., food processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.