Enzyme Mechanisms: Wedging open a catalytic site

The activation mechanism of the nitric oxide receptor has been revealed by cryo-electron microscopy.
  1. Annie Beuve  Is a corresponding author
  1. Rutgers, the State University of New Jersey, United States

Of the many ways to regulate blood pressure, the simplest is to dilate or narrow the blood vessels. In the body, the gas nitric oxide (NO) binds to the enzyme soluble guanylyl cyclase (sGC) to relax blood vessels and decrease blood pressure. When the interaction between NO and sGC is disrupted, people develop hypertension and pulmonary arterial hypertension, and have a higher risk of heart failure. This makes sGC a major target for the treatment of cardiovascular diseases.

Drugs that activate the sGC enzyme have been used for many years, often without understanding their mechanism of action. One striking example is nitroglycerin, which has been used to treat angina pectoris since the end of the 19th century. It took over 100 years to discover that nitroglycerin and other nitrates work by generating NO or its derivatives, which then stimulate sGC to produce cyclic guanosine monophosphate (cGMP). The cGMP produced in this way relaxes the coronary arteries and increases blood flow to the heart. Unfortunately, nitroglycerin and most NO donors induce resistance, meaning that the patient becomes less and less responsive to ever-increasing doses of the drug.

In 1994, a small molecule called YC-1, which is not an NO derivative, was found to dramatically enhance the ability of the sGC enzyme to produce cGMP at low NO concentrations (Ko et al., 1994; Friebe et al., 1998). In healthy individuals, NO is produced by endothelial cells, which line the inside of blood vessels. When these cells stop performing their normal roles, the resulting low levels of NO lead to vascular diseases.

Nowadays, pulmonary arterial hypertension is treated with molecules similar to YC-1 (such as Adempas), which can stimulate the sGC enzyme without inducing resistance. These drugs are also in clinical trials for the treatment of chronic heart failure and have the potential to be used for treating chronic kidney diseases, hypertension and fibrotic diseases (Buys et al., 2018; Sandner et al., 2018). Two questions have been the subject of intense scrutiny and conflicting data: where does YC-1 bind to sGC, and how does it increase the impact of NO. Now, in eLife, Michael Marletta, Jim Hurley and colleagues at Berkeley, including Ben Horst and Adam Yokom as joint first authors, report the results of cryo-electron microscopy (cryo-EM) and small angle X-ray scattering experiments that shed light on these questions (Horst et al., 2019).

Structurally and kinetically, sGC is a complex enzyme. It is a heterodimer formed by two subunits, α and β, which each contain four domains: an N-terminal heme nitric oxide/oxygen binding (HNOX) domain; a Per/Arnt/Sim (PAS)-fold domain; a coiled-coil (CC) domain; and a catalytic domain at the C-terminal (Figure 1A). NO binds to the heme in the β subunit. Over the years, X-ray structures of each domain have been obtained, but attempts to determine the full-length 3D structure of the sGC enzyme have failed repeatedly. Without such structures it is not clear how these domains rearrange upon NO binding to create the most favorable conformation for the production of cGMP (Montfort et al., 2017; Horst and Marletta, 2018). Moreover, it was impossible to determine how molecules similar to YC-1 achieve their therapeutic effect.

Soluble guanylyl cyclase (sGC) and the treatment of pulmonary arterial hypertension (PAH).

(A) The enzyme sGC contains two subunits, α and β, and each subunit is composed of four domains: the N-terminal heme nitric oxide/oxygen binding (HNOX) domain, the Per-Arnt-Sim (PAS) domain, the coiled-coil (CC) domain, and the catalytic domain. (B) In pulmonary arterial hypertension blood vessels contract excessively, thus increasing arterial pressure (left); this process can be reversed (blue arrow) by nitric oxide (NO, hexagons) and drugs called small sGC stimulators (triangles). These molecules bind to sGC and change its conformation so it becomes active and can catalyze the conversion of guanosine triphosphate (GTP, green hexagons) to cyclic guanosine monophosphate (cGMP, light green circles). This last molecule acts a secondary messenger and induces the dilation of blood vessels. Horst et al. have shed light on the mechanism of activation for the sGC enzyme. In the inactive state (bottom), the catalytic site is inaccessible, so the enzyme cannot catalyze the reaction that converts GTP to cGMP; there is also a sharp bend in each coiled-coil domain. When NO binds to the heme in the HNOX domain of the β subunit, the HNOX and PAS domains rotate by 71° leading to the straightening of the bend in the two coiled-coil domains, which opens the catalytic site, thus allowing the enzyme to catalyze the conversion of GTP to cGMP (top). Small stimulators such as YC-1 bind into the space created between the β HNOX and CC domains, acting as a wedge that keeps the enzyme active for longer.

Horst et al. employed cryo-EM to obtain the full-length 3D structures of sGC in a non-activated state and in an activated state after the addition of NO and YC-1. Comparing these two structures reveals the structural changes caused by NO binding and suggest a likely mechanism for the therapeutic action of YC-1-like drugs. When NO binds to the sGC enzyme, a major rearrangement of the HNOX and PAS domains in the β subunit takes place, inducing the CC domains, which include a bend, to become straighter. This twist, in turn, creates an interface between the HNOX and CC domains in the β subunit. These conformational changes lead to opening of the catalytic pocket, making it possible for the sGC enzyme to bind guanosine triphosphate (GTP) and catalyze the production of cGMP (Figure 1B, right panel). Interestingly, YC-1 was found in a site created by the NO-induced rearrangement of the β HNOX and CC domains, where it could act as a wedge to maintain an unbent active conformation.

The structures reported by Horst et al. are similar to recent cryo-EM structures of sGC with and without excess NO (Kang et al., 2019). This suggests that activation at low NO concentrations enhanced by YC-1 is structurally comparable to that at high NO concentrations in the absence of YC-1. These models of sGC activation end years of frustration in the field, and provide a structural basis to further improve the design of small sGC stimulators that could be used to treat cardiovascular diseases (Figure 1).

It remains to be resolved whether YC-1 binds to the same site in the absence of NO, since there are a number of other possible sites (Stasch et al., 2001; Lamothe et al., 2004; Wales et al., 2018). In the current study, small-angle X-ray scattering models indicate that, in the absence of YC-1, sGC enzymes with an NO-equivalent molecule bound to the heme are a mix of bent-inactive and partially extended-activated structures. However, it is still unclear how higher concentrations of NO induce the conversion to the fully extended-activated structure. The mechanism of deactivation also remains unknown. Resolving these two matters is a priority for researchers working on the physiological role of NO.

References

Article and author information

Author details

  1. Annie Beuve

    Annie Beuve is at the Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers, the State University of New Jersey, Newark, United States

    For correspondence
    beuveav@njms.rutgers.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4389-5388

Publication history

  1. Version of Record published: November 8, 2019 (version 1)

Copyright

© 2019, Beuve

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.

Metrics

  • 699
    Page views
  • 60
    Downloads
  • 2
    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. Annie Beuve
(2019)
Enzyme Mechanisms: Wedging open a catalytic site
eLife 8:e52418.
https://doi.org/10.7554/eLife.52418

Further reading

    1. Biochemistry and Chemical Biology
    Kanwal Kayastha et al.
    Research Article

    Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD+ binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    Laura M Doherty et al.
    Research Article

    Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).