Biology subjects

Karska, N.

Publications and source records attributed to Karska, N..

2 recordsLinked to original sources

bioRxiv2026

Membrane-dependent structural organization of cowpox virus CPXV012 and its recognition of TAP

Cowpox virus CPXV012 inhibits MHC class I antigen presentation by interfering with TAP-dependent peptide transport, but its membrane-dependent structural organization and dynamic behavior remain incompletely defined. Here, we investigated the conformational properties of CPXV012 in membrane-mimicking environments and in a model of the CPXV012-TAP complex. CPXV012 was divided into three peptide constructs corresponding to the N-terminal cytosolic region, transmembrane segment, and C-terminal ER-luminal domain. The peptides were analyzed by circular dichroism spectroscopy, multidimensional NMR spectroscopy, and molecular dynamics simulations, and the resulting structural information was integrated into a full-length CPXV012 model. CD spectra showed that CPX-E1 and CPX-C2 are predominantly disordered in aqueous solution but acquire ordered, mainly -helical features in DPC micelles. NMR analysis in DPC-d38 micelles provided residue-level assignments and structural restraints supporting restrained structure calculations for both peptides. In three independent 1 {micro}s molecular dynamics simulations of the CPXV012-TAP complex, CPXV012 preserved a reproducible two-helical organization. The N-terminal/transmembrane region behaved as a relatively stable structural element, whereas the ER-luminal segment showed greater local flexibility. Interface analysis indicated that CPXV012 contacts both TAP1 and TAP2, with recurrent interactions concentrated in the luminal Y47-I69 region and involving polar and charge-complementary contacts. These results support a model in which membrane-associated structuring positions CPXV012 for TAP recognition, while the flexible ER-luminal region forms the main TAP-interacting surface. This structural framework complements existing functional models of CPXV012-mediated TAP inhibition.

biochemistry↗

bioRxiv2026

Unlocking viral evasion: Luminal charge interactions in BoHV-1 UL49.5 allosterically control TAP degradation

The UL49.5 protein of bovine alphaherpesvirus 1 (BoHV-1) is known to inhibit the transporter associated with antigen processing (TAP) and interfere with antigen presentation, in part by promoting TAP degradation. However, the role of electrostatic interactions within the N-terminal luminal domain in controlling these processes remains unclear. Here, we combined circular dichroism (CD), solution nuclear magnetic resonance spectroscopy (NMR), all-atom molecular dynamics simulations, and cell-based assays to define the structural and functional contribution of charged residues within the N-terminal luminal domain of UL49.5. Two N-terminal variants of UL49.5, UL49.522-56RR(30-31)DD and UL49.522-56D36K, with substitutions of charged-reversal amino acid residues, were designed. These two mutants formed membrane-induced -helical structures but showed altered helix stability and interaction patterns. Molecular dynamics simulations of the UL49.5-TAP complexes revealed that wild-type UL49.5 forms a stable electrostatic interface with TAP, particularly in the unkinked conformation, while charge-reversal mutations remodel salt-bridge networks, destabilize the luminal helix, and alter the positioning and dynamics of the transmembrane and cytoplasmic C-terminal regions. The structural changes within the N-terminus alter the exposure of the C-terminal degron required for KLHDC3-dependent degradation. Consistent with these findings, the mutants did not induce proteasomal degradation of TAP, despite maintaining near wild-type levels of downregulation of MHC class I. Together, these results identify N-terminal electrostatic interactions as allosteric determinants of UL49.5-driven TAP degradation and demonstrate that TAP degradation can be mechanically uncoupled from downregulation of MHC class I. This study improves our understanding of viral immune evasion strategies and potential therapeutic targets.

biochemistry↗