Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics

Abstract

A B S T R A C TThe transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far.Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 -encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembraneintracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer.Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structurebased insight in its mechanism of action which might be helpful in designing new drugs against viral infections.

Investigation of the Effects of Primary Structure Modifications within the RRE Motif on the Conformation of Synthetic Bovine Herpesvirus 1‐Encoded UL49.5 Protein Fragments

“…In biological studies, it has been observed that the deletion of these three amino acid residues in the protein leads to a loss of transporting properties. We found the RRE residues in an α‐helix (fragment R9–A19 in the native protein) of our structural model, [11] and it is likely that the presence of this helix plays a role in the inhibition of antigenic peptide transport through the TAP channel. To verify this hypothesis, we designed and synthesized three peptide analogs with a modified RRE sequence.…”

“…This means that the studied peptides adopt one dominant conformation in the (1–27) fragment, while the C‐terminal fragment of the analogs is characterized by greater mobility. The increased mobility of the C‐terminal fragment (as in the case of the native BoHV peptide [11] ) may result from the presence of two proline residues in positions 31 and 32 or from cis‐trans isomerism. The chemical shifts of the protons of these residues in the studied analogs are very similar and their difference does not exceed ±0.25 ppm.…”

“…By analyzing the structure of the analog compared to the structure of the native peptide (wt), [11] it can be seen that the E11G and RRE(9–11)AAA peptides are characterized by a higher content of the helical structure in both micellar solutions in comparison to the native fragment, whereas the RRE(9–11)GGG analog is characterized by a lower content of helical structure. The presence of secondary structures (calculated with CDPro software using the CONTINLL SMP56 algorithm [5,16] ) indicates that the peptides adopt a structure that is α‐helical and disordered ( Figure S1 ).…”

“…Due to its orientation in the membrane, three biologically relevant fragments of the protein can be distinguished: the N‐terminal part (N.BHV), located on the side of the endoplasmic reticulum lumen or extracellular; the transmembrane domain (TM.BHV); and the C‐terminal fragment (C.BHV) on the cytoplasmic side [10] . In our previous study, [11] we determined the experimental structure of the BoHV‐1 UL49.5 protein using nuclear magnetic resonance (NMR) and molecular dynamics (MD) technology in the cell membrane environment. Our results revealed that UL49.5 adopted a highly flexible membrane‐proximal helical structure in the extracellular fragment.…”

Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

“…Designing an optimal fluorescent TAP construct was hampered by the lack of complete structural information about TAP-UL49.5 interaction, and thus, it required an experimental evaluation of different TAP-GFP variants. The latest structural study on BoHV-1 UL49.5 revealed its 3D structure, while subsequent molecular docking experiments proposed three different possible orientations of TAP-UL49.5 complex in which UL49.5 was suggested to interact simultaneously with both TAP subunits [44]. However, these models were predicted based on the structure of ICP47-arrested TAP conformation [10], and therefore the actual UL45.9-TAP binding model needs to be further confirmed.…”