Role of the Coiled-Coil Domain in Allosteric Activity Regulation in Soluble Guanylate Cyclase.

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in higher eukaryotes, including humans. NO-dependent signaling via sGC is associated with important physiological effects in the vascular, pulmonary, and neurological systems, and sGC itself is an established drug target for the treatment of pulmonary hypertension due to its central role in vasodilation. Despite isolation in the late 1970s, high-resolution structural information on full-length sGC remained elusive until recent cryo-electron microscopy structures were determined of the protein in both the basal unactivated state and the NO-activated state. These structures revealed large-scale conformational changes upon activation that appear to be centered on rearrangements within the coiled-coil (CC) domains in the enzyme. Here, a structure-guided approach was used to engineer constitutively unactivated and constitutively activated sGC variants through mutagenesis of the CC domains. These results demonstrate that the activation-induced conformational change in the CC domains is necessary and sufficient for determining the level of sGC activity.

Corrole-protein interactions in H-NOX and HasA.

Replacing the native porphyrin cofactor in haem proteins has led to the development of novel designer proteins for a variety of applications. In most cases, haem analogues bind in a way that is comparable to the iron porphyrin, but this is not necessarily the case for complexes bearing non-exchangeable ligands. This study probes how a P[double bond, length as m-dash]O corrole binds to functionally disparate hemoproteins: a haem-dependent oxygen sensor (H-NOX) and a haem-scavenging protein (HasA). The results demonstrate that the protein-cofactor interactions are distinct from the native, haem-bound holoprotein. In H-NOX, the P[double bond, length as m-dash]O unit primarily hydrogen bonds with the haem-ligating histidine (H102), rather than the hydrogen-bonding network that stabilises the Fe(ii)-O2 complex in the native protein. In the absence of H102, the protein is still able to bind the corrole, albeit at reduced levels. Molecular dynamics simulations were utilised to determine the flexibility of apo H-NOX and revealed the coupled motion of key residues necessary for corrole binding. In the case of HasA, the P[double bond, length as m-dash]O unit does not primarily interact with either the haem-ligating histidine (H32) or tyrosine (Y75). Instead, histidine 83, the hydrogen-bonding partner for Y75, is critical for P[double bond, length as m-dash]O corrole binding. The conformation of HasA is interrogated by site-specifically labelling the protein and exploiting Förster resonance energy transfer (FRET) to determine the dye-cofactor distance. HasA reconstituted with the P[double bond, length as m-dash]O corrole exhibits an extended, apo-like conformation. Together, these results demonstrate that non-natural cofactors can bind to proteins in unexpected ways and highlight the need to uncover these interactions for the further development of designer haem proteins.

Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation.

The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo-electron microscopy (cryo-EM) enabled visualization of full-length sGC for the first time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation.

Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH3+), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH3+, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster. The A-cluster is largely buried and is inaccessible to CFeSP for methylation. Here we use electron microscopy to capture multiple snapshots of ACS that reveal previously uncharacterized domain motion, forming extended and hyperextended structural states. In these structural states, the A-cluster is accessible for methylation by CFeSP.

The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase.

Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO2 using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners. These enzymes tend to be extremely O2-sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from Desulfovibrio vulgaris (Dv) appears unique: exposure to micromolar concentrations of O2 on the minutes-time scale only reversibly inhibits the enzyme, and full activity is recovered after reduction. Here, we examine whether this unusual property of Dv CODH results from the nature of its D-cluster, which is a [2Fe-2S] cluster, instead of the [4Fe-4S] cluster observed in all other characterized CODHs. To this aim, we produced and characterized a Dv CODH variant where the [2Fe-2S] D-cluster is replaced with a [4Fe-4S] D-cluster through mutagenesis of the D-cluster-binding sequence motif. We determined the crystal structure of this CODH variant to 1.83-Å resolution and confirmed the incorporation of a [4Fe-4S] D-cluster. We show that upon long-term O2-exposure, the [4Fe-4S] D-cluster degrades, whereas the [2Fe-2S] D-cluster remains intact. Crystal structures of the Dv CODH variant exposed to O2 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under aerobic conditions retains 30% activity relative to a fully anaerobic purification, compared to 10% for the variant, and the WT enzyme loses activity more slowly than the variant upon prolonged aerobic storage. The D-cluster is therefore a key site of irreversible oxidative damage in Dv CODH, and the presence of a [2Fe-2S] D-cluster contributes to the O2-tolerance of this enzyme. Together, these results relate O2-sensitivity with the details of the protein structure in this family of enzymes.

Crystallographic Characterization of the Carbonylated A-Cluster in Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Wood-Ljungdahl pathway allows for autotrophic bacterial growth on carbon dioxide, with the last step in acetyl-CoA synthesis catalyzed by the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). ACS uses a complex Ni-Fe-S metallocluster termed the A-cluster to assemble acetyl-CoA from carbon monoxide, a methyl moiety and coenzyme A. Here, we report the crystal structure of CODH/ACS from Moorella thermoacetica with substrate carbon monoxide bound at the A-cluster, a state previously uncharacterized by crystallography. Direct structural characterization of this state highlights the role of second sphere residues and conformational dynamics in acetyl-CoA assembly, the biological equivalent of the Monsanto process.

Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase.

The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel-iron-sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2 Like other complex metalloenzymes, CODH requires dedicated assembly machinery to form the fully intact and functional C-cluster. In particular, nickel incorporation into the C-cluster depends on the maturation factor CooC; however, the mechanism of nickel insertion remains poorly understood. Here, we compare X-ray structures (1.50-2.48 Å resolution) of CODH from Desulfovibrio vulgaris (DvCODH) heterologously expressed in either the absence (DvCODH-CooC) or presence (DvCODH+CooC) of co-expressed CooC. We find that the C-cluster of DvCODH-CooC is fully loaded with iron but does not contain any nickel. Interestingly, the so-called unique iron ion (Feu) occupies both its canonical site (80% occupancy) and the nickel site (20% occupancy), with addition of reductant causing further mismetallation of the nickel site (60% iron occupancy). We also demonstrate that a DvCODH variant that lacks a surface-accessible iron-sulfur cluster (the D-cluster) has a C-cluster that is also replete in iron but lacks nickel, despite co-expression with CooC. In this variant, all Feu is in its canonical location, and the nickel site is empty. This D-cluster-deficient CODH is inactive despite attempts to reconstitute it with nickel. Taken together, these results suggest that an empty nickel site is not sufficient for nickel incorporation. Based on our findings, we propose a model for C-cluster assembly that requires both CooC and a functioning D-cluster, involves precise redox-state control, and includes a two-step nickel-binding process.

Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase.

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity.

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator.

Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Šresolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ∼33 Šacross an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within the active site that play likely roles in facilitating catalysis, leading to the proposal of a modified mechanistic scheme involving two distinct roles for the active site histidine. We also identify putative substrate entrance and product egress routes within the enzyme, which are discussed in the context of previously reported biochemical observations. Our structure lays a foundation for further biochemical and structural characterization of PhaC, which could assist in engineering efforts for the production of eco-friendly materials.

Waltzing around cofactors.

The metallocofactor involved in fixing nitrogen is not a rigid scaffold, as was previously thought.