IRAA: A statistical tool for investigating a protein-protein interaction interface from multiple structures.

Understanding protein-protein interactions (PPIs) is fundamental to infer how different molecular systems work. A major component to model molecular recognition is the buried surface area (BSA), that is, the area that becomes inaccessible to solvent upon complex formation. To date, many attempts tried to connect BSA to molecular recognition principles, and in particular, to the underlying binding affinity. However, the most popular approach to calculate BSA is to use a single (or in some cases few) bound structures, consequently neglecting a wealth of structural information of the interacting proteins derived from ensembles corresponding to their unbound and bound states. Moreover, the most popular method inherently assumes the component proteins to bind as rigid entities. To address the above shortcomings, we developed a Monte Carlo method-based Interface Residue Assessment Algorithm (IRAA), to calculate a combined distribution of BSA for a given complex. Further, we apply our algorithm to human ACE2 and SARS-CoV-2 Spike protein complex, a system of prime importance. Results show a much broader distribution of BSA compared to that obtained from only the bound structure or structures and extended residue members of the interface with implications to the underlying biomolecular recognition. We derive that specific interface residues of ACE2 and of S-protein are consistently highly flexible, whereas other residues systematically show minor conformational variations. In effect, IRAA facilitates the use of all available structural data for any biomolecular complex of interest, extracting quantitative parameters with statistical significance, thereby providing a deeper biophysical understanding of the molecular system under investigation.

An Electrostatically-steered Conformational Selection Mechanism Promotes SARS-CoV-2 Spike Protein Variation.

After two years since the outbreak, the COVID-19 pandemic remains a global public health emergency. SARS-CoV-2 variants with substitutions on the spike (S) protein emerge increasing the risk of immune evasion and cross-species transmission. Here, we analyzed the evolution of the S protein as recorded in 276,712 samples collected before the start of vaccination efforts. Our analysis shows that most variants destabilize the S protein trimer, increase its conformational heterogeneity and improve the odds of the recognition by the host cell receptor. Most frequent substitutions promote overall hydrophobicity by replacing charged amino acids, reducing stabilizing local interactions in the unbound S protein trimer. Moreover, our results identify "forbidden" regions that rarely show any sequence variation, and which are related to conformational changes occurring upon fusion. These results are significant for understanding the structure and function of SARS-CoV-2 related proteins which is a critical step in vaccine development and epidemiological surveillance.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

Insights on cross-species transmission of SARS-CoV-2 from structural modeling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 31 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic and confirmed cases of cross-species transmission, the question of the extent to which this transmission is possible emerges, as well as what molecular features distinguish susceptible from non-susceptible animal species. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also allow us to predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.

Structural models of human ACE2 variants with SARS-CoV-2 Spike protein for structure-based drug design.

Emergence of coronaviruses poses a threat to global health and economy. The current outbreak of SARS-CoV-2 has infected more than 28,000,000 people and killed more than 915,000. To date, there is no treatment for coronavirus infections, making the development of therapies to prevent future epidemics of paramount importance. To this end, we collected information regarding naturally-occurring variants of the Angiotensin-converting enzyme 2 (ACE2), an epithelial receptor that both SARS-CoV and SARS-CoV-2 use to enter the host cells. We built 242 structural models of variants of human ACE2 bound to the receptor binding domain (RBD) of the SARS-CoV-2 surface spike glycoprotein (S protein) and refined their interfaces with HADDOCK. Our dataset includes 140 variants of human ACE2 representing missense mutations found in genome-wide studies, 39 mutants with reported effects on the recognition of the RBD, and 63 predictions after computational alanine scanning mutagenesis of ACE2-RBD interface residues. This dataset will help accelerate the design of therapeutics against SARS-CoV-2, as well as contribute to prevention of possible future coronaviruses outbreaks.

Structure, biochemical and kinetic properties of recombinant Pst2p from Saccharomyces cerevisiae, a FMN-dependent NAD(P)H:quinone oxidoreductase.

The genome of the yeast Saccharomyces cerevisiae encodes four flavodoxin-like proteins, namely Lot6p, Pst2p, Rfs1p and Ycp4p. Thus far only Lot6p was characterized in detail demonstrating that the enzyme possesses NAD(P)H:quinone oxidoreductase activity. In the present study, we heterologously expressed PST2 in Escherichia coli and purified the produced protein to conduct a detailed biochemical and structural characterization. Determination of the three-dimensional structure by X-ray crystallography revealed that Pst2p adopts the flavodoxin-like fold and forms tetramers independent of cofactor binding. The lack of electron density for FMN indicated weak binding, which was confirmed by further biochemical analysis yielding a dissociation constant of 20±1µM. The redox potential of FMN bound to Pst2p was determined to -89±3mV and is thus 119mV more positive than that of free FMN indicating that reduced FMN binds ca. five orders of magnitude tighter to Pst2p than oxidized FMN. Due to this rather positive redox potential Pst2p is unable to reduce free FMN or azo dyes as reported for other members of the flavodoxin-like protein family. On the other hand, Pst2p efficiently catalyzes the NAD(P)H dependent two-electron reduction of natural and artificial quinones. The kinetic mechanism follows a ping-pong bi-bi reaction scheme. In vivo experiments with a PST2 knock out and overexpressing strain demonstrated that Pst2p enables yeast cells to cope with quinone-induced damage suggesting a role of the enzyme in managing oxidative stress.