4BM7

Crystal Structure of IgG Fc F241A mutant with native glycosylation

Summary for 4BM7

• Entry DOI: 10.2210/pdb4bm7/pdb
• Descriptor: IG GAMMA-1 CHAIN C REGION, 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[beta-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: immune system, glycan
• Biological source: HOMO SAPIENS (HUMAN)
• Total number of polymer chains: 2
• Total formula weight: 53854.99

Authors

Yu, X.,Baruah, K.,Harvey, D.J.,Vasiljevic, S.,Alonzi, D.S.,Song, B.,Higgins, M.K.,Bowden, T.A.,Crispin, M.,Scanlan, C.N. (deposition date: 2013-05-07, release date: 2013-07-03, Last modification date: 2024-10-16)

Primary citation

Yu, X.,Baruah, K.,Harvey, D.J.,Vasiljevic, S.,Alonzi, D.S.,Song, B.,Higgins, M.K.,Bowden, T.A.,Scanlan, C.N.,Crispin, M.
Engineering Hydrophobic Protein-Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies.
J.Am.Chem.Soc., 135:9723-, 2013

PubMed Abstract: Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

PubMed: 23745692
DOI: 10.1021/JA4014375

Experimental method

X-RAY DIFFRACTION (1.95 Å)