Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

Human interleukin 5 receptor alpha (IL5Ralpha) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokine-recognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5Ralpha to define the residues involved in IL5 recognition. Soluble forms of IL5Ralpha variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp(55), Asp(56), and Glu(58)) and D2 (Lys(186) and Arg(188)) domains, but also in the D3 (Arg(297)) domain. Modeling of the tertiary structure of IL5Ralpha indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5 x IL5Ralpha system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface

The Case for S2: The Potential Benefits of the S2 Subunit of the SARS-CoV-2 Spike Protein as an Immunogen in Fighting the COVID-19 Pandemic

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.Fil: Shah, Priyanka. Drexel University; Estados UnidosFil: Canziani, Gabriela Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Carter, Erik P.. Drexel University; Estados UnidosFil: Chaiken, Irwin. Drexel University; Estados Unido

Altered Env conformational dynamics as a mechanism of resistance to peptide‑triazole HIV‑1 inactivators.

Background: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimerpost-print4093 K

Macromolecular biorecognition : principles and methods/ Edit.: Irwin Chaiken

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