The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

• Published in: Journal of molecular biology Vol. 435; no. 5; p. 167964
• Main Authors: Graul, Małgorzata, Karska, Natalia, Wąchalska, Magda, Krupa, Paweł, Ślusarz, Magdalena J., Lubocki, Marcin, Bieńkowska-Szewczyk, Krystyna, Rodziewicz-Motowidło, Sylwia, Sieradzan, Adam K., Lipińska, Andrea D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.03.2023
• Subjects: Amino Acid Motifs; Antigen Presentation; herpesvirus; Herpesvirus 1, Bovine - immunology; Membrane Transport Proteins - metabolism; MHC class I; molecular dynamics; Proline - chemistry; Proline - genetics; Protein Transport; TAP transporter; UL49.5; BoHV-1; TAP; FL; PLC; CTLs; MHC; NBD; HSV-1; UNRES; herpesvirus; MD; MJS; cryo-EM; aa; MOI; IP; MHC class I; RMSD; TMD; molecular dynamics; ER; TAP transporter; UL49.5; ERAD; PAA; PE; TM; SASA
• DOI: 10.1016/j.jmb.2023.167964
• ISSN: 0022-2836

[Display omitted] •UL49.5 of BoHV-1 inhibits TAP transporter by mechanisms undetermined on a molecular level.•Mutations in 52PPQ54 proline hinge resulted in impaired TAP binding and inhibition.•According to MD, PPQ membrane anchor is key for UL49.5 structure and dynamical behavior.•52PPQ54 is required for the putative C-terminal degron accessibility and TAP degradation.•Our findings advance structural understanding of the UL49.5 protein and its functional regions. Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.