X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

Abstract

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion

“…Coordinates for ordered N-linked glycans from PDB: 5T3X and PDB: 5VN3 Env structures ( Gristick et al., 2016 , Ozorowski et al., 2017 ) were fit separately as rigid bodies at potential N-linked glycosylation sites (PNGSs) at which EM density was apparent ( Figure S4 B), and glycan rings outside of EM density were removed. The entire complex was refined using phenix.real_space_refine with secondary structure restraints for protein and geometric restraints for protein and N-glycan residues ( Adams et al., 2010 , Afonine et al., 2018 , Agirre et al., 2015 , Gristick et al., 2017 ) ( Table S1 ).…”

“…β-strand restraints were applied to strands A-D and V2 throughout refinement in both Coot and Phenix. The entire complex was refined using phenix.real_space_refine with secondary structure restraints for protein and geometric restraints for protein and N-glycan residues ( Adams et al., 2010 , Afonine et al., 2018 , Agirre et al., 2015 , Gristick et al., 2017 ) ( Table S2 ).…”

Automatically Fixing Errors in Glycoprotein Structures with Rosetta

“…We identified four incorrect anomeric configurations and 23 high-energy ring conformations using the Privateer software ( Agirre et al., 2015b ) and detected by visual inspection that one structure was missing a glycosidic bond ( Table 1 ). We compared refinement of our Rosetta-based method with that of Phenix refinement alone and, for models with high-energy ring conformations in the input, Phenix refinement with constraints generated by Privateer ( Agirre, 2017b , Gristick et al., 2017 ). Following refinement of these 12 structures with our Phenix-Rosetta protein structure refinement pipeline, which alternates real- and reciprocal-space refinement, we were able to markedly improve the carbohydrate geometry, as assessed by Privateer.…”

“…This Rosetta glycan refinement protocol expands upon previous iterations ( Labonte et al., 2017 ) by avoiding fitting stereochemically unfavorable glycan structures into sparse experimental data and instead yielding physically realistic geometries based on prior knowledge of saccharide chemical properties. Using a benchmark set of 12 deposited crystal structures, we demonstrated our algorithm is capable of correcting models containing significant errors in glycan geometry ( Table 1 ) above and beyond previous methods ( Agirre, 2017b , Gristick et al., 2017 ). This is likely due to the increased radius of convergence of our refinement, as well as the ability to flip anomeric state.…”

“…The approach builds upon previous Rosetta-based structure determination tools guided by low-resolution experimental data ( Frenz et al., 2017 , Wang et al., 2015 ) and makes use of a previously developed framework for modeling of carbohydrates ( Labonte et al., 2017 ). Compared with previous glycan refinement methods ( Agirre, 2017b , Gristick et al., 2017 ), our approach (1) uses a physically realistic force field to ensure glycan geometry remains correct even under large conformational changes, and (2) adds the ability to change the anomer of the glycan. These protocols are publicly available with the latest Rosetta release and enable refinement of glycoprotein structures against cryoEM ( Wang et al., 2016 ) and X-ray crystallography data, the latter taking advantage of the combined Phenix-Rosetta reciprocal-space refinement pipeline ( Terwilliger et al., 2012 ).…”