Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites.

The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins (i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2_NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating.

A novel mechanism for ATP to enhance the functional oligomerization of TDP-43 by specific binding.

Pathological TDP-43 aggregation has been found in ∼98% ALS and other neurodegenerative diseases including Alzheimer's. TDP-43 N-terminal domain (NTD) was recently shown to form a tubular super-helical filament by oligomerization in vivo, which functions to prevent its pathological aggregation. ATP, the universal energy currency with very high concentrations in all living cells, was recently decoded to act as a biological hydrotrope to maintain protein homeostasis. Here by NMR spectroscopy, we reveal for the first time that at physiological concentrations ATP binds the TDP-43 NTD to enhance its oligomerization. Most strikingly, this binding is specifically coupled with oligomerization because three mutants with the capacity of oligomerization eliminated lose the ability to bind ATP. Our study thus provides a novel mechanism for ATP to prevent pathological aggregation by specific binding; and further implies that ATP might have many previously-unknown functions in cells by binding to proteins other than the classic ATP-dependent proteins/enzymes.

Oligomerization of Retrovirus Integrases.

Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.

Insights into the structural peculiarities of the N-terminal and receptor binding domains of the spike protein from the SARS-CoV-2 Omicron variant.

Since the new variant of SARS-CoV-2, Omicron (BA.1) has raised serious concerns, it is important to investigate the effects of mutations in the NTD and RBD domains of the spike protein for the development of COVID-19 vaccines. In this study, computational analysis of the Wuhan and Omicron NTDs and RBDs in their unbound and bound states to mAb 4A8 and ACE2 were performed. In addition, the interaction of NTD with antibody and RBD with ACE2 were evaluated in the presence of long glycans. The results show that long glycans at the surface of NTDs can reduce the accessibility of protein epitopes, thereby reducing binding efficiency and neutralizing potency of specific antibodies. Also, our findings indicate that the existence of the long glycans result in increased stability and enhanced affinity of the RBD to ACE2 in the Wuhan and Omicron variant. Key residues that play an important role in increasing the structural stability of the protein were identified using RIN analysis and in the state of interaction with mAb 4A8 and ACE2 through per-residue decomposition analysis. Further, the results of the free energy binding calculation using MM/GBSA method show that the Omicron variant has a higher infectivity than the Wuhan. This study provides a better understanding of the structural changes in the spike protein and can be useful for the development of novel therapeutics.

The Spike Protein of SARS-coV2 19B (S) Clade Mirrors Critical Features of Viral Adaptation and Coevolution.

Pathogens including viruses evolve in tandem with diversity in their animal and human hosts. For SARS-coV2, the focus is generally for understanding such coevolution on the virus spike protein, since it demonstrates high mutation rates compared to other genome regions, particularly in the receptor-binding domain (RBD). Viral sequences of the SARS-coV2 19B (S) clade and variants of concern from different continents were investigated, with a focus on the A.29 lineage, which presented with different mutational patterns within the 19B (S) lineages in order to learn more about how SARS-coV2 may have evolved and adapted to widely diverse populations globally. Results indicated that SARS-coV2 went through evolutionary constrains and intense selective pressure, particularly in Africa. This was manifested in a departure from neutrality with excess nonsynonymous mutations and a negative Tajima D consistent with rapid expansion and directional selection as well as deletion and deletion-frameshifts in the N-terminal domain (NTD region) of the spike protein. In conclusion, we hypothesize that viral transmission during epidemics through populations of diverse genomic structures and marked complexity may be a significant factor for the virus to acquire distinct patterns of mutations within these populations in order to ensure its survival and fitness, explaining the emergence of novel variants and strains.

First report of computational protein-ligand docking to evaluate susceptibility to HIV integrase inhibitors in HIV-infected Iranian patients.

Background: Iran has recently included integrase (INT) inhibitors (INTIs) in the first-line treatment regimen in human immunodeficiency virus (HIV)-infected patients. However, there is no bioinformatics data to elaborate the impact of resistance-associated mutations (RAMs) and naturally occurring polymorphisms (NOPs) on INTIs treatment outcome in Iranian patients. Method: In this cross-sectional survey, 850 HIV-1-infected patients enrolled; of them, 78 samples had successful sequencing results for INT gene. Several analyses were performed including docking screening, genotypic resistance, secondary/tertiary structures, post-translational modification (PTM), immune epitopes, etc. Result: The average docking energy (E value) of different samples with elvitegravir (EVG) and raltegravir (RAL) was more than other INTIs. Phylogenetic tree analysis and Stanford HIV Subtyping program revealed HIV-1 CRF35-AD was the predominant subtype (94.9%) in our cases; in any event, online subtyping tools confirmed A1 as the most frequent subtype. For the first time, CRF-01B and BF were identified as new subtypes in Iran. Decreased CD4 count was associated with several factors: poor or unstable adherence, naïve treatment, and drug user status. Conclusion: As the first bioinformatic report on HIV-integrase from Iran, this study indicates that EVG and RAL are the optimal INTIs in first-line antiretroviral therapy (ART) in Iranian patients. Some conserved motifs and specific amino acids in INT-protein binding sites have characterized that mutation(s) in them may disrupt INT-drugs interaction and cause a significant loss in susceptibility to INTIs. Good adherence, treatment of naïve patients, and monitoring injection drug users are fundamental factors to control HIV infection in Iran effectively.

A Routine Sanger Sequencing Target Specific Mutation Assay for SARS-CoV-2 Variants of Concern and Interest.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have a potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein, along with certain mutations in other regions within the S protein as specific variants, in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.

Structural Comparison of the SARS CoV 2 Spike Protein Relative to Other Human-Infecting Coronaviruses.

Coronaviruses (CoV) are enveloped positive-stranded RNA viruses and, historically, there are seven known human-infecting CoVs with varying degrees of virulence. CoV attachment to the host is the first step of viral pathogenesis and mainly relies on the spike glycoprotein located on the viral surface. Among the human-infecting CoVs, only the infection of SARS CoV 2 (SARS2) among humans resulted to a pandemic which would suggest that the protein structural conformation of SARS2 spike protein is distinct as compared to other human-infecting CoVs. Surprisingly, the possible differences and similarities in the protein structural conformation between the various human-infecting CoV spike proteins have not been fully elucidated. In this study, we utilized a computational approach to generate models and analyze the seven human-infecting CoV spike proteins, namely: HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1, SARS CoV, MERS CoV, and SARS2. Model quality assessment of all CoV models generated, structural superimposition of the whole protein model and selected S1 domains (S1-CTD and S1-NTD), and structural comparison based on RMSD values, Tm scores, and contact mapping were all performed. We found that the structural orientation of S1-CTD is a potential structural feature associated to both the CoV phylogenetic cluster and lineage. Moreover, we observed that spike models in the same phylogenetic cluster or lineage could potentially have similar protein structure. Additionally, we established that there are potentially three distinct S1-CTD orientation (Pattern I, Pattern II, Pattern III) among the human-infecting CoVs. Furthermore, we postulate that human-infecting CoVs in the same phylogenetic cluster may have similar S1-CTD and S1-NTD structural orientation. Taken together, we propose that the SARS2 spike S1-CTD follows a Pattern III orientation which has a higher degree of similarity with SARS1 and some degree of similarity with both OC43 and HKU1 which coincidentally are in the same phylogenetic cluster and lineage, whereas, the SARS2 spike S1-NTD has some degree of similarity among human-infecting CoVs that are either in the same phylogenetic cluster or lineage.

Crystal structure of the S1 subunit N-terminal domain from DcCoV UAE-HKU23 spike protein.

The DcCoV UAE-HKU23 coronavirus is a newly-found betacoronavirus (betaCoV) that can infect human cells. The viral spike protein plays pivotal roles in mediating receptor-recognition and membrane-fusion, and is therefore a key factor involved in viral pathogenesis and inter-species transmission. Here we reported the structural and functional characterization of the spike N-terminal domain (NTD) from DcCoV UAE-HKU23 (HKU23-NTD). Via mucin-binding assays, we showed that HKU23-NTD is able to bind sugars. We further solved the structure of HKU23-NTD, performed structure-guided mutagenesis and successfully located the potential sugar-binding pockets in the structure. Furthermore, via comparison of available betaCoV NTD structures, we demonstrated that betaCoV NTDs contain a conserved ß-sandwich core, but exhibit variant folds in the peripheral elements located in the top-ceiling region and on the lateral side. While showing different compositions and structures, these peripheral elements are topologically equivalent ß-sandwich-core insertions, highlighting a divergent evolution process for betaCoVs to form different lineages.