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.