Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-.ANG. resolution

Abstract

The model of human Fc fragment was refined at 2.9-A resolution. Two different automated procedures for crystallographic refinement were used [Deisenhofer, J., & Steigemann, W. (1975) Acta Crystallogr., Sec. B B31, 238; Jack, A., & Levitt, M. (1978) Acta Crystallogr., Sect. A A34, 931]. The final R value is 0.22. The dimer of CH3 domains closely resembles the CH1-CL aggregate in Fab fragments. There is no contact between CH2 domains. The contact between CH2 and CH3 domains has about one-third of the size of the CH3-CH3 contact. The carbohydrate, a branched chain of nine hexose units, covers parts of the C-contact face of the CH2 domain, shielding hydrophobic residues on this surface. Six atoms of the carbohydrate are within hydrogen-bonding distance of atoms in the CH2 domain. Crystallographic refinement of the complex between Fc fragment and fragment B of protein A from Staphylococcus aureus reduced the R value of the model is 0.24. A major part of the structure of fragment B consists of two alpha helics; the rest of the polypeptide chain is folded irregularly. In the crystal, fragment B forms two contacts with Fc fragment molecules. Contact 1 involves residues from both helices of fragment B, and residues from the CH2 and CH3 domains of FC, and is predominantly hydrophobic. Contact 2 is smaller than contact 1. Residues from the second helix and adjacent residues of fragment B and residues only from the CH3 domain of Fc contribute to contact 2. The nature of contact 2 is mainly polar and includes a sulfate ion. There are strong arguments that contact 1 is the fragment B-Fc contact formed in solution under physiological conditions, while contact 2 is a crystal contact.

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

“…Though this appears to be a somewhat convoluted mechanism to explain the observations, it has been shown that there is contact between the two glycans through the two core mannose residues and that in the absence of this interaction the structure of the CH 2 domains are perturbed, highlighting the interaction between the glycans and this region of the Fc. [24][25][26] Now we consider the possibility that the effect observed was due to oxidation of Met430. Residues HC[247-253] have been shaded dark red in Figure 3, it can be seen that in the crystal structure these residues form an a-helix, which is located adjacent to methionines 253 and 430.…”

Endoglycosidase treatment abrogates IgG arthritogenicity: Importance of IgG glycosylation in arthritis

“…IgG molecules are mainly glycosylated at Asn-297 of the CH2 domain within the Fc region [3,4], with variable galactosylation but limited sialylation. The remaining glycosylation occurs in the hypervariable regions of the Fab region, with the position and frequency of occurrence being dependent on the presence of the consensus sequence Asn-Xaa-Thr/ Ser for N-glycosylation, and is characterized by a high incidence of sialylated structures.…”

“…X-Ray crystal electron density maps of the IgG-Fc revealed that the N297-linked glycan is sequestered within the internal space enclosed by the CH2 domains. There are extensive non-covalent interactions between the carbohydrate and the protein moiety, resulting in reciprocal influences on conformation [3].…”

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

“…As assessed by Protein A binding, antibody titers ranged from 0 to 982 mg/L. However, because the CH2 and CH3 domains form the binding interface with Protein A, 22 we anticipated that some antibody variants carrying aldehyde tags in these regions would be expressed but undetected by our Protein A-coated ForteBio sensor used for titer measurements. Accordingly, scanned regions of the CH2 domain where antibody titers were low as measured by Protein A (e.g., 188V through 206V) were tested for antibody titers by ForteBio using a Protein L-coated sensor.…”