Evolutionary trajectory of receptor binding specificity and promiscuity of the spike protein of SARS-CoV-2.

SARS-CoV-2 infects cells by attachment to its receptor-the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation-, and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affects the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism and capacity to evade immune responses during evolution.

The key role of E418 carboxyl group in the formation of Nt.BspD6I nickase active site: Structural and functional properties of Nt.BspD6I E418A mutant.

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.

Heme interacts with c1q and inhibits the classical complement pathway.

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.

Kinetics and thermodynamics of interaction of coagulation factor VIII with a pathogenic human antibody.

Replacement therapy in hemophilia A with exogenous coagulation factor VIII (FVIII) often results in the development of FVIII-neutralizing antibodies, referred to as inhibitors. Despite of large number of studies on the functional properties of FVIII inhibitors, detailed physicochemical characterization of their interactions is not available. Here we studied the biophysical mechanism of the interaction between a human pathogenic antibody--BO2C11 and its target antigen--FVIII. Kinetic and thermodynamic analyses implied that this interaction is not accompanied by significant conformational changes in the proteins. The data also suggested that association of BO2C11 to FVIII is driven mainly by a hydrophobic effect. The protein electrostatics however played a decisive role in this association. Thus, a gradual increase in ionic strength resulted in a considerable increase in the association rate of binding of BO2C11 to FVIII. Such an ionic strength-dependency is uncommon for other antibody-antigen interactions. Our data suggest that electrostatic effects observed for BO2C11-FVIII association may arise from high-energy penalty of desolvation of the charged residues at the binding interfaces. We hypothesize that untypical ionic strength dependence of association of BO2C11 to FVIII reflects the nature of the recognized epitope, namely a molecular surface involved in the binding of FVIII to phospholipids. The presented data provide mechanistic information about FVIII neutralization by an inhibitory antibody and also contribute to the understanding of the general mechanisms of antibody-antigen interactions.

PHEMTO: protein pH-dependent electric moment tools.

PHEMTO (protein pH-dependent electric moment tools) is released in response to the high demand in protein science community for evaluation of electrostatic characteristics in relations to molecular recognition. PHEMTO will serve protein scientists with new advanced features for analysis of protein molecular interactions: Electric/dipole moments, their pH-dependence and in silico charge mutagenesis effects on these properties as well as alternative algorithms for electric/dipole moment computation--Singular value decomposition of electrostatic potential (EP) to account for reaction field. The implementation is based on long-term experience--PHEI mean field electrostatics and PHEPS server for evaluation of global and local pH-dependent properties. However, PHEMTO is not just an update of our PHEPS server. Besides standard electrostatics, we offer new, advanced and useful features for analysis of protein molecular interactions. In addition our algorithms are very fast. Special emphasis is given to the interface--intuitive and user-friendly. The input is comprised of the atomic coordinate file in Protein Data Bank format. The advanced user is provided with a special input section for addition of non-polypeptide charges. The output covers actually full electrostatic characteristics but special emphasis is given to electric/dipole moments and their interactive visualization. PHEMTO server can be accessed at http://phemto.orgchm.bas.bg/.

PHEPS: web-based pH-dependent Protein Electrostatics Server.

PHEPS (pH-dependent Protein Electrostatics Server) is a web service for fast prediction and experiment planning support, as well as for correlation and analysis of experimentally obtained results, reflecting charge-dependent phenomena in globular proteins. Its implementation is based on long-term experience (PHEI package) and the need to explain measured physicochemical characteristics at the level of protein atomic structure. The approach is semi-empirical and based on a mean field scheme for description and evaluation of global and local pH-dependent electrostatic properties: protein proton binding; ionic sites proton population; free energy electrostatic term; ionic groups proton affinities (pK(a,i)) and their Coulomb interaction with whole charge multipole; electrostatic potential of whole molecule at fixed pH and pH-dependent local electrostatic potentials at user-defined set of points. The speed of calculation is based on fast determination of distance-dependent pair charge-charge interactions as empirical three exponential function that covers charge-charge, charge-dipole and dipole-dipole contributions. After atomic coordinates input, all standard parameters are used as defaults to facilitate non-experienced users. Special attention was given to interactive addition of non-polypeptide charges, extra ionizable groups with intrinsic pK(a)s or fixed ions. The output information is given as plain-text, readable by 'RasMol', 'Origin' and the like. The PHEPS server is accessible at http://pheps.orgchm.bas.bg/home.html.

Role of Ca2+ in the electrostatic stability and the functional activity of the globular domain of human C1q.

C1q is the recognition subunit of the classical pathway of the complement system and a major connecting link between classical pathway-driven innate immunity and IgG- or IgM-mediated acquired immunity. The basic structural subunit of C1q is composed of an N-terminal triple-helical collagen-like region and a C-terminal heterotrimeric globular head domain (gC1q) that is made up of individual A, B, and C chains. Recent crystallographic studies have revealed that the gC1q domain, which is the main target-binding region of C1q, has a compact and spherical heterotrimeric assembly, held together by both electrostatic and nonpolar interactions, with quasi-3-fold symmetry. A characteristic feature of the gC1q domain is the presence of a exposed Ca(2+) located near the apex. We have investigated, using theoretical and experimental approaches, the role of Ca(2+) in the electrostatic stability and target-binding properties of the native C1q as well as recombinant monomeric forms of the C-terminal regions of the A, B, and C chains. Here, we report that Ca(2+) primarily influences the target recognition properties of C1q toward IgG, IgM, C-reactive protein, and pentraxin 3. At pH 7.4, the loss of Ca(2+) leads to changes in the direction of electric moment from coaxial (where the putative C-reactive protein-binding site is located) to perpendicular to the molecular axis (toward the most likely IgG-binding site), which appears important for target recognition by C1q and subsequent complement activation.

Mutational analyses of the recombinant globular regions of human C1q A, B, and C chains suggest an essential role for arginine and histidine residues in the C1q-IgG interaction.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.