Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization.

Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerization, and we used pressure-jump NMR spectroscopy to measure long-range NOE contacts that exist exclusively in the oligomeric NMR-invisible state. The kinetics of oligomerization and dissociation were probed at the residue-specific level, revealing that the oligomerization process is initiated in the C-terminal half of the segment. Using pressure-jump NMR, the degree of order in the oligomer at the sites of Pro residues was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the oligomeric state. NOE contacts and secondary 13C chemical shifts in the oligomeric state indicate the presence of an 11-residue α-helix, preceded by a small intramolecular antiparallel ß-sheet, with slower formation of long-range intermolecular interactions to N-terminal residues. Although none of the models generated by AlphaFold2 for the amelotin monomer was consistent with experimental data, subunits of a hexamer generated by AlphaFold-Multimer satisfied intramolecular NOE and chemical shift data and may provide a starting point for developing atomic models for the oligomeric state.

SARS-CoV-2 omicron variants harbor spike protein mutations responsible for their attenuated fusogenic phenotype.

Since the emergence of the Omicron variants at the end of 2021, they quickly became the dominant variants globally. The Omicron variants may be more easily transmitted compared to the earlier Wuhan and the other variants. In this study, we aimed to elucidate mechanisms of the altered infectivity associated with the Omicron variants. We systemically evaluated mutations located in the S2 sequence of spike and identified mutations that are responsible for altered viral fusion. We demonstrated that mutations near the S1/S2 cleavage site decrease S1/S2 cleavage, resulting in reduced fusogenicity. Mutations in the HR1 and other S2 sequences also affect cell-cell fusion. Based on nuclear magnetic resonance (NMR) studies and in silico modeling, these mutations affect fusogenicity possibly at multiple steps of the viral fusion. Our findings reveal that the Omicron variants have accumulated mutations that contribute to reduced syncytial formation and hence an attenuated pathogenicity.

Real-time Exchange of the Lipid-bound Intermediate and Post-fusion States of the HIV-1 gp41 Ectodomain.

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38 °C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal mol-1. The temperature dependence of the kinetics indicates that the folding occurs through a high-energy transition state which may resemble a 5HB structure. The energetics and kinetics of gp41 folding in the context of membrane bilayers provide a molecular basis for an improved understanding of viral membrane fusion.

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.

Advances in NMR Spectroscopy of Weakly Aligned Biomolecular Systems.

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.

Transient lipid-bound states of spike protein heptad repeats provide insights into SARS-CoV-2 membrane fusion.

Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the intervening structural transition, important for the design of fusion inhibitors, has remained elusive. Using nuclear magnetic resonance spectroscopy and other biophysical methods, we demonstrate the presence of α-helical, membrane-bound, intermediate states of spike's heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface while in a slow dynamic equilibrium with the postfusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.

Concentration-Dependent Structural Transition of the HIV-1 gp41 MPER Peptide into α-Helical Trimers.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings.

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. 15N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

DRB4 dsRBD1 drives dsRNA recognition in Arabidopsis thaliana tasi/siRNA pathway.

In Arabidopsis thaliana, endogenous trans-acting and exogenous siRNA pathways are initiated by the interaction of DRB4 with trigger dsRNA. Further, DCL4:DRB4 complex cleaves the dsRNA into 21 bp siRNA. Understanding molecular determinants and mechanistic details of dsRNA recognition by DRB4 is vital for inducing long-term RNAi-mediated gene regulation in plants. Here, we present solution structures of individual and concatenated DRB4 dsRBDs and demonstrate modes of dsRNA binding by employing NMR, ITC and site-specific mutagenesis. While both dsRBDs adopt the canonical α-ß-ß-ß-α fold, key structural differences and ms-µs dynamics located at the RNA binding region were observed for dsRBD1. These features favor dsRBD1 to orient itself and make stronger tripartite contact with dsRNA, a feature missing in dsRBD2. Additionally, the inter-domain orientation induced by the linker restricts the mobility of dsRBD2, resulting in the steric hindrance of α1 helix in dsRBD2, and leads in further reduction of its dsRNA binding activity. Our study deciphers functional roles of DRB4 domains by showing that dsRBD1 drives the tasiRNA/siRNA pathway. Furthermore, we identify a potential role of the C-terminal region of DRB4 in protein:protein interaction as it possesses six PxxP motifs, binds to Zn2+ and contains a small structural domain.

Recent excitements in protein NMR: Large proteins and biologically relevant dynamics.

The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecular NMR spectroscopists to overcome the size limitation barrier (approx. 20 kDa) in de novo structure determination of proteins. The utility of these techniques was immediately demonstrated on large proteins and protein complexes (e.g. GroELGroES, ClpP protease, Hsp90-p53, 20S proteasome, etc.). Further, recent methodological developments such as Residual Dipolar Couplings and Paramagnetic Relaxation Enhancement allowed accurate measurement of long-range structural restraints. Additionally, Carr-Purcell-Meiboom-Gill (CPMG), rotating frame relaxation experiments (R1(rho)) and saturation transfer experiments (CEST and DEST) created never-before accessibility to the (mu)s-ms timescale dynamic parameters that led to the deeper understanding of biological processes. Meanwhile, the excitement in the field continued with a series of developments in the fast data acquisition methods allowing rapid structural studies on less stable proteins. This review aims to discuss important developments in the field of biomolecular NMR spectroscopy in the recent past, i.e., in the post TROSY era. These developments not only gave access to the structural studies of large protein assemblies, but also revolutionized tools in the arsenal of today's biomolecular NMR and point to a bright future of biomolecular NMR spectroscopy.

Chemical shift assignments of DRB4 (1-153), a dsRNA binding protein in A. thaliana RNAi pathway.

RNA interference (RNAi) is a conserved biological response to dsRNA and regulates the expression of protein-coding genes to mediate resistance to both endogenous parasitic and exogenous pathogenic nucleic acids. In RNAi pathway, dsRNA binding proteins assists Dicer at various stages of RNAi. In plants, DRB4, is a multidomain protein containing two dsRNA binding domains that recognizes the long exogenous/endogenous dsRNA and presents it to Ribonuclease enzyme, Dicer like 4, resulting in the production of 21 nt small interfering RNA. Here, we report nearly complete backbone and sidechain chemical shift assignments of N-terminus of DRB4 (1-153, ~18 kDa), containing both double stranded RNA binding domains and the linker.

Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.

The association of RDE-4 (RNAi defective 4), a protein containing two dsRBDs (dsRNA-binding domains), with long dsRNA and Dcr-1 (Dicer1 homologue) initiates the siRNA pathway in Caenorhabditis elegans. Unlike its homologues in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to co-operativity. The linker and dsRBD2 are indispensable for RDE-4's simultaneous interaction with dsRNA and Dcr-1. In the present study, we have determined the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, both dsRBDs of RDE-4 show modified structural features such as truncation in the ß1-ß2 loop that rationalize RDE-4's relatively weak dsRNA affinity. Structure and binding studies demonstrate that dsRBD2 plays a decisive role in the RDE-4-dsRNA interaction; however, in contrast with previous findings, we found ephemeral interaction of RDE-4 dsRBD1 with dsRNA. More importantly, mutations in two tandem lysine residues (Lys217 and Lys218) in dsRBD2 impair RDE-4's dsRNA-binding ability and could obliterate RNAi initiation in C. elegans. Additionally, we postulate a structural basis for the minimal requirement of linker and dsRBD2 for RDE-4's association with dsRNA and Dcr-1.

Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1-243), an RNA interference initiation protein in C. elegans.

The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.