Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches

Abstract

The glycans of the major capsid protein (Vp54) of chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein.

Chloroviruses

“…However, it is now known that it exists as a trimer in the viral capsomers with a size of ~162 kD. [51] and C is from Yan et al [52]. All are published with permission.…”

“…Some of their most distinctive features include: (i) the four glycoforms share a common core structure; (ii) The glycoforms are related to the non-stochiometric presence of two monosaccharides, L-arabinose and D-mannose; (iii) The most abundant glycoform consists of nine neutral monosaccharide residues organized in a highly-branched fashion ( Figure 3); (iv) None of the N-linked glycans are attached to a typical Asn-X-(Thr/Ser) consensus site in the protein; (v) The glycans are attached to the protein by a β-glucose linkage, which is rare in nature and has only been reported in glycoproteins from a few organisms [54][55][56][57]; (vi) The glycoform contains a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue and two rhamnose residues with opposite configurations. [51] and (C) is from Yan et al [52]. All are published with permission.…”

“…The second region extends the conserved core with other monosaccharides, which are specific for each chlorovirus [58]. A detailed description of the complete structure of the PBCV-1 MCP was recently described by combining crystallographic data with molecular modeling (Figure 2A) [51]. This is one of the first glycoproteins, if not the first, to have its complete structure resolved.…”

Biophysical Approaches to Solve the Structures of the Complex Glycan Shield of Chloroviruses

“…However, glycosylation of the PBCV-1 major capsid protein differs from the scenario described above because the virus encodes most, if not all, of the machinery for the process (Van Etten et al 2010. Solving the structure of the four Vp54 N-linked glycans (De Castro et al 2013) proved the uniqueness of PBCV-1 glycosylation: (1) Vp54 has four Asn-linked glycans, and none of the Asn are located in an Asn-X-(Thr/ Ser) sequon characteristic of ER-located glycosyltransferases (Nandhagopal et al 2002;De Castro et al 2018); (2) the glycans are attached to Asn by a β-glucose linkage, which is rare in nature; (3) the glycans are highly branched and consist of eight to ten neutral monosaccharides (Fig. 2); (4) the four glycoforms contain a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue, and two rhamnose residues with opposite absolute configurations; (5) the four glycoforms differ by the nonstoichiometric presence of two monosaccharides, l-arabinose and d-mannose; and (6) there is a core region (Fig.…”

Viral glycoproteomes: technologies for characterization and outlook for vaccine design

“…Additional changes to protein design are sometimes made to increase solubility and homogeneity, bringing it even further away from the native glycoprotein . Combining the different techniques compensates for the shortcomings of the individual methods and provides a more complete picture of viral glycan structure and presentation, particularly when dealing with unusual glycan structures . Structural investigations are very useful in determining the positions of glycans within the tertiary or quaternary structures of proteins, as well as glycan‐dependent interactions with lectins and antibodies , and attempts have been made to engineer vaccine formulations based on altering the glycan shield density of HIV‐1, with little success in virus neutralization by the immune sera .…”