It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with ...cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.
The human macrophage migration inhibitory factor (MIF) superfamily consists of MIF and D‐dopachrome tautomerase (also called MIF‐2). Both MIF proteins have critical innate and adaptive ...immunomodulatory functions in regulating differentiation, expression of pro‐inflammatory cytokines, and activation and recruitment of immune cells. They are broadly and constitutively expressed, but elevated levels of MIF family proteins are upregulated in various inflammatory diseases such as asthma, acute respiratory distress syndrome (ARDS) and arthritis, and antibody neutralization of MIF decreased inflammatory symptoms in murine models. While their potential as drug targets has led to the creation of a few commercial inhibitors of MIF, the efficacy of targeting MIF proteins as a therapeutic is currently obstructed by a lack of molecular level detail about their structures and dynamics and how those properties impact disease‐state activity.
Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. The MIF homolog DDT (MIF‐2) has low sequence identity with MIF (35%) though they overlap in multiple functions, including activation of the CD74 receptor, and share a nearly identical tertiary structure. This prompted the question of whether this dynamic regulatory network is conserved within the superfamily. We identify a comparable allosteric network in MIF‐2, connecting the catalytic site to CD74‐activating residues, showing with solution nuclear magnetic resonance (NMR) spectroscopy that dynamic communication is preserved in MIF‐2 despite differences in primary sequence. Disruption of the allosteric relay by mutagenesis also attenuates MIF‐2 enzymatic activity in vitro and biological CD74 signaling in vivo, highlighting amino acids in this pathway as trigger residues for potential functional control of the protein.
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an ...intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.
Macrophage migration inhibitory factor (MIF) is an immunomodulatory protein with a pathogenic activity in various inflammatory disorders, autoimmune diseases, and cancer. The majority of ...MIF-triggered pathological conditions are associated with activation of the cell surface receptor CD74. In the absence of small molecule antagonists that directly target CD74, MIF variants and MIF-ligand complexes have served as modulators of CD74 activity. These molecules have been reported to have either antagonistic or agonistic effects against the receptor, although the mechanistic parameters that distinguish the two groups are largely unknown. Through molecular dynamics simulations and NMR experiments, we explored the relationship between MIF’s catalytically active N-terminus and the surface residues important for the activation of CD74. We found that the two sites are connected via backbone dynamics that are propagated to the CD74 activation surface of MIF, from the β2 and β4 strands. Our results also provide mechanistic evidence that explain the functional characteristics of MIF variants, serving as CD74 agonists or antagonists. Such findings are of high importance for understanding the MIF-induced activation of CD74 as well as for the development of highly potent CD74 therapeutics.
Remdesivir is an adenosine analogue that has a cyano substitution in the C1′ position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1′ atom with its ...strong electron-withdrawing cyano group. Within the replication–transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.
Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize ...these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.
A hallmark of tightly regulated high-fidelity enzymes is that they become activated only after encountering cognate substrates, often by an induced-fit mechanism rather than conformational selection. ...Upon analysis of molecular dynamics trajectories, we recently discovered that the Cas9 HNH domain exists in three conformations: 1) Y836 (which is two residues away from the catalytic D839 and H840 residues) is hydrogen bonded to the D829 backbone amide, 2) Y836 is hydrogen bonded to the backbone amide of D861 (which is one residue away from the third catalytic residue N863), and 3) Y836 is not hydrogen bonded to either residue. Each of the three conformers differs from the active state of HNH. The conversion between the inactive and active states involves a local unfolding-refolding process that displaces the Cα and side chain of the catalytic N863 residue by ∼5 Å and ∼10 Å, respectively. In this study, we report the two largest principal components of coordinate variance of the HNH domain throughout molecular dynamics trajectories to establish the interconversion pathways of these conformations. We show that conformation 2 is an obligate step between conformations 1 and 3, which are not directly interconvertible without conformation 2. The loss of hydrogen bonding of the Y836 side chain in conformation 3 likely plays an essential role in activation during local unfolding-refolding of an α-helix containing the catalytic N863. Three single Lys-to-Ala mutants appear to eliminate this substrate-independent activation pathway of the wild-type HNH nuclease, thereby enhancing the fidelity of HNH cleavage.
MIF is a ubiquitous protein involved in proinflammatory processes, which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid, produced by ...neutrophil-released myeloperoxidase (MPO) under inflammatory conditions, effectively oxidizes MIF into the oxMIF isoform, which is specifically recognized by the anti-oxMIF therapeutic antibody, ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure, including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation, whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA, SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor, CD74, which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor, and a route for therapeutic intervention targeting the oxMIF isoform.
D-Dopachrome tautomerase (D-DT; or MIF-2) is a multifunctional protein with immunomodulatory properties and a documented pathogenic role in inflammation and cancer that is associated with activation ...of the cell surface receptor CD74. Alongside D-DT, macrophage migration inhibitory factor (MIF) is also known to activate CD74, promoting pathogenesis. While the role of the MIF/CD74 axis has been extensively studied in various disease models, the late discovery of the D-DT/CD74 axis has led to a poor investigation into the D-DT-induced activation mechanism of CD74. A previous study has identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the first selective and reversible inhibitor of D-DT and reported its potency to block the D-DT-induced activation of CD74 in a cell-based model. In this study, we employ molecular dynamics simulations and nuclear magnetic resonance experiments to study 4-CPPC-induced changes to the dynamic profile of D-DT. We found that binding of the inhibitor remarkably promotes the conformational flexibility of C-terminal without impacting the structural stability of the biological assembly. Consequently, long-range intrasubunit (>11 Å) and intersubunit (>30 Å) communications are enabled between distal regions. Communication across the three subunits is accomplished via 4-CPPC, which serves as a communication bridge after Val113 is displaced from its hydrophobic pocket. This previously unrecognized structural property of D-DT is not shared with its human homolog, MIF, which exhibits an impressive C-terminal rigidity even in the presence of an inhibitor. Considering the previously reported role of MIF’s C-terminal in the activation of CD74, our results break new ground for understanding the functionality of D-DT in health and disease.
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