Structural basis for HCMV Pentamer recognition by antibodies and neuropilin 2

Abstract

Human cytomegalovirus (HCMV) encodes for multiple surface glycoproteins and glycoprotein complexes. One of these complexes, the HCMV Pentamer (gH, gL, UL128, UL130 and UL131), mediates tropism to both epithelial and endothelial cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here we describe the cryo-EM structure of NRP2 bound to the HCMV Pentamer. The high-affinity interaction between these proteins is calcium-dependent and differs from the canonical C-terminal arginine (CendR) binding that NRP2 typically utilizes. The interaction is primarily mediated by NRP2 domains a2 and b2, which interact with UL128 and UL131. We also determine the structures of four human-derived neutralizing antibodies in complex with the HCMV Pentamer to define susceptible epitopes. The two most potent antibodies recognize a novel epitope yet do not compete with NRP2 binding. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization, and serve as a guide for the development of HCMV treatments and vaccines.

Structural basis for HCMV Pentamer receptor recognition and antibody neutralization

“…We compared our cryo-EM structure of Pentamer bound to NRP2 with the previously determined crystal structure of Pentamer in the absence of a receptor [Protein Data Bank (PDB): 5VOB; ( 15 )] and the structure of the HCMV gHgLgO Trimer [gH and gL subunits only; ( 16 )]. Notably, and in agreement with previous findings ( 12 ), very similar conformations of gH/gL are observed across all comparisons (fig. S4, A and B), implying that a different mechanism must underlie the activation of the HCMV fusion machinery, as discussed below.…”

“…Three main sites of interaction between NRP2 and the Pentamer were identified, in agreement with recent observations ( Fig. 1, E to G ) ( 12 ). At site 1, a calcium-coordinating region in the a2 domain of NRP2 establishes charge interactions with residues localized in the N-terminal domain of UL128 of the Pentamer ( Fig.…”

“…1D). This region was poorly resolved in our cryo-EM map, suggesting that a1 domain binding to monomeric Pentamer is dynamic, consistent with the finding that NRP2 is still able to interact with Pentamer in the absence of the a1 domain, albeit to a lower degree (12).…”

“…1, E, F, and H , and fig. S3A) ( 12 ). Upon Pentamer binding, the NRP2 a1 domain is displaced from the a2b1b2 core and repositioned at the site of attachment between UL128 and gL ( Fig.…”

“…NRP1 and NRP2 proteins are known to interact with several common partners, including vascular endothelial growth factor (VEGF) and the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), through a mode in which their respective b1 domains interact with a [R/K]XX[R/K] (CendR) motif in the binding partner (13,14). However, our structure and studies from the McLellan's group (12) unambiguously demonstrate that the interaction between Pentamer and NRP2 does not involve this common binding surface on the b1 domain but rather requires specific interactions between the a2 and b2 domains of NRP2 and the UL components of the Pentamer, and in particular UL128 and UL131A (Fig. 1, E to G, and fig.…”