Structure and enzymatic properties of human retroviral-like aspartic protease 1 and functional roles of disease-associated mutations.

PROTEASE AND ITS MODULATION BY SMALL MOLECULES”

HIV-1 Nef Homodimerization as a Structural Mechanism for Kinase Activation and Small Molecule Inhibitor Action

The HIV-1 accessory protein Nef plays a central role in viral pathogenesis by enhancing viral replication, modulating cellular signaling, and evading immune recognition, making it a compelling target for therapeutic intervention. Nef lacks enzymatic activity and instead functions through diverse interactions with host cell proteins including the Src-family tyrosine kinase, Hck. Kinase activation may requires Nef homodimerization, as mutations disrupting the Nef dimer interface impair kinase activation as well as many other Nef functions. In the present study, we investigated the structural consequences of dimer interface mutations and their impact on Nef interactions with Hck regulatory domains. Using size-exclusion chromatography, multi-angle light scattering and crystallography, we found that mutations at dimer interface residues Leu112 and Phe121 abolish recombinant Nef protein dimerization while preserving the overall Nef fold, resulting in monomeric 1:1 complexes with Hck SH3 or SH3-SH2 domain proteins. These findings demonstrate that the broad phenotypic effects of interface mutations arise from loss of Nef dimerization rather than global misfolding or perturbation of SH3 binding. We also investigated the effects of small molecule Nef inhibitors on homodimer formation. These compounds, like the dimerization-defective mutations, suppress kinase activation, viral replication and restore immune recognition of HIV-infected cells. Using a SplitFAST fluorescence complementation assay, we provide direct evidence that these inhibitors disrupt Nef homodimer formation in solution. Co-crystallization of a wild-type Nef:SH3 complex with an inhibitor also prevented homodimer formation. Computational docking identified a shared pocket for six active Nef inhibitors formed by the Nef dimer interface but lost in the monomer. Together, our findings support homodimerization as a structural feature essential for many Nef functions and validate disruption of this interface as a promising therapeutic strategy against HIV-1.

SV40 Large T antigen inhibits the host serine protease FAM111A through a zinc-dependent, cleavage-avoiding mechanism

SV40 Large T antigen (LT) is essential for viral replication and a key determinant of host range. This host-range function is mediated by the C-terminal domain (LT-C) through binding to the host serine protease FAM111A, but the underlying mechanism has remained unclear. Here, we report the X-ray crystal structure of the FAM111A serine protease domain in complex with LT-C, revealing the structural basis for direct inhibition of FAM111A. LT-C uses a previously unrecognized zinc-binding motif and a P1-like phenylalanine residue to engage the FAM111A active site through a substrate-mimicking mechanism while avoiding proteolytic cleavage and covalent complex formation. Mutations disrupting either feature abolish FAM111A inhibition and impair SV40 propagation in cells. Consistent with this mechanism, SV40 host restriction requires FAM111A protease activity, which must be antagonized by LT-C for productive infection. Together, these findings define a zinc-dependent, cleavage-avoiding mechanism of protease inhibition that highlights an evolutionary arms race between SV40 and host antiviral proteases.

Helicolysins emerge as a new family of metzincin metallopeptidases in host-pathogen interactions.

The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.

Structural Features of a Tiny Viral Protein, ORF7b of SARS-CoV-2

Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure–function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of −4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation.

Insights into the enzyme kinetics, structural features, and in silico inhibition of Leishmania donovani prolidase.

Prolidase is a specialized metalloprotease that overcomes the resistance of proline-containing peptide bonds to proteolysis by exclusively hydrolyzing dipeptides with a C-terminal proline. Despite its potential significance in the life cycle of protozoan parasites, knowledge of prolidase in these organisms remains limited. In this study, the complete coding sequence of Leishmania donovani prolidase (LdProl) was cloned into an expression vector. The recombinant protein was purified using affinity chromatography, and its molecular weight was assessed by size-exclusion chromatography. The purified LdProl exhibited enzymatic activity with a strong substrate preference for Trp-Pro-pNA, and manganese ions were identified as essential cofactors for optimal activity. LdProl also stimulated innate immune mechanisms through elevating pro-inflammatory cytokines and nitric oxide levels, suggesting a role in macrophage-mediated immunomodulation. Fluorescence spectroscopy revealed pH-dependent conformational changes, particularly under strongly acidic conditions, whereas circular dichroism spectroscopy indicated a predominantly α-helical structure that was most stable at neutral pH. Computational modeling demonstrated a conserved three-dimensional structure, metal-binding site, and stable dimeric interface similar to prolidases from other organisms. Docking studies identified Trp-Pro-pNA as the substrate with the highest binding affinity, and molecular dynamics simulations confirmed the formation of stable enzyme-substrate complexes. Virtual screening yielded two lead compounds with higher binding affinities for LdProl than for its human counterpart, along with favorable ADME properties. Molecular dynamics simulations further validated these compounds by demonstrating the stability of enzyme-inhibitor complexes. Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.