The H163A mutation unravels an oxidized conformation of the SARS-CoV-2 main protease.

The main protease of SARS-CoV-2 (Mpro) is an important target for developing COVID-19 therapeutics. Recent work has highlighted Mpro's susceptibility to undergo redox-associated conformational changes in response to cellular and immune-system-induced oxidation. Despite structural evidence indicating large-scale rearrangements upon oxidation, the mechanisms of conformational change and its functional consequences are poorly understood. Here, we present the crystal structure of an Mpro point mutant (H163A) that shows an oxidized conformation with the catalytic cysteine in a disulfide bond. We hypothesize that Mpro adopts this conformation under oxidative stress to protect against over-oxidation. Our metadynamics simulations illustrate a potential mechanism by which H163 modulates this transition and suggest that this equilibrium exists in the wild type enzyme. We show that other point mutations also significantly shift the equilibrium towards this state by altering conformational free energies. Unique avenues of SARS-CoV-2 research can be explored by understanding how H163 modulates this equilibrium.

Biochemical, structural, and kinetic characterization of PPi -dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii.

Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.

A metal-dependent conformational change provides a structural basis for the inhibition of CTP synthase by gemcitabine-5'-triphosphate.

CTP synthases (CTPS) catalyze the de novo production of CTP using UTP, ATP, and l-glutamine with the anticancer drug metabolite gemcitabine-5'-triphosphate (dF-dCTP) being one of its most potent nucleotide inhibitors. To delineate the structural origins of this inhibition, we solved the structures of Escherichia coli CTPS (ecCTPS) in complex with CTP (2.0 Å), 2'-ribo-F-dCTP (2.0 Å), 2'-arabino-F-CTP (2.4 Å), dF-dCTP (2.3 Å), dF-dCTP and ADP (2.1 Å), and dF-dCTP and ATP (2.1 Å). These structures revealed that the increased binding affinities observed for inhibitors bearing the 2'-F-arabino group (dF-dCTP and F-araCTP), relative to CTP and F-dCTP, arise from interactions between the inhibitor's fluorine atom exploiting a conserved hydrophobic pocket formed by F227 and an interdigitating loop from an adjacent subunit (Q114-V115-I116). Intriguingly, crystal structures of ecCTPS•dF-dCTP complexes in the presence of select monovalent and divalent cations demonstrated that the in crystallo tetrameric assembly of wild-type ecCTPS was induced into a conformation similar to inhibitory ecCTPS filaments solely through the binding of Na+ -, Mg2+ -, or Mn2+ •dF-dCTP. However, in the presence of potassium, the dF-dCTP-bound structure is demetalated and in the low-affinity, non-filamentous conformation, like the conformation seen when bound to CTP and the other nucleotide analogues. Additionally, CTP can also induce the filament-competent conformation linked to high-affinity dF-dCTP binding in the presence of high concentrations of Mg2+ . This metal-dependent, compacted CTP pocket conformation therefore furnishes the binding environment responsible for the tight binding of dF-dCTP and provides insights for further inhibitor design.

Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase.

During our efforts to characterize the regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for phosphoenolpyruvate (PEP) becomes reduced several days after cell lysis. A 1.8 Å crystallographic structure of L-PYK with the S12D mimic of phosphorylation indicates that Cys436 is oxidized, the first potential insight into explaining the effect of "aging". Interestingly, the oxidation is only to sulfenic acid despite the crystal growth time period of 2 weeks. Mutagenesis confirms that the side chain of residue 436 is energetically coupled to PEP binding. Mass spectrometry confirms that the oxidation is present in solution and is not an artifact caused by X-ray exposure. Exposure of the L-PYK mutations to H2O2 also confirms that PEP affinity is sensitive to the nature of the side chain at position 436. A 1.95 Å structure of the C436M mutant of L-PYK, the only mutation at position 436 that has been shown to strengthen PEP affinity, revealed that the methionine substitution results in the ordering of several N-terminal residues that have not been ordered in previous structures. This result allowed speculation that oxidation of Cys436 and phosphorylation of the N-terminus at Ser12 may function through a similar mechanism, namely the interruption of an activating interaction between the nonphosphorylated N-terminus with the nonoxidized main body of the protein. Mutant cycles were used to provide evidence that mutations of Cys436 are energetically synergistic with N-terminal modifications, a result that is consistent with phosphorylation of the N-terminus and oxidation of Cys436 functioning through mechanisms with common features. Alanine-scanning mutagenesis was used to confirm that the newly ordered N-terminal residues were important to the regulation of enzyme function by the N-terminus of the enzyme (i.e., not an artifact caused by the introduced methionine substitution) and to further define which residues in the N-terminus are energetically coupled to PEP affinity. Collectively, these studies indicate energetic coupling (and potentially mechanistic similarities) between the oxidation of Cys436 and phosphorylation of Ser12 in the N-terminus of L-PYK.

The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis.

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded ß sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded ß sheet MinE dimer with the released ß strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.

Determination of the structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC.

The three Min proteins spatially regulate Z ring positioning in Escherichia coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP dependence of MinC and MinE binding to MinD.

Structural insights into the mechanism of PEPCK catalysis.

Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.