Structural characterization of a human Fc fragment engineered for lack of effector functions

Abstract

The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S ('TM') causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 A using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236-445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc.

Figures

Figure 1

(a) Representation of the contents of the asymmetric unit of the Fc/TM crystal. The only visible mutation comprising ‘TM’, P331S, is indicated in red. One zinc ion is shown chelated by two spatially close histidine residues. The conventional ‘horseshoe’-shaped homodimeric Fc fragment would be achieved by invoking a crystallographic twofold symmetry operator. The carbohydrate residues attached to Asn297 were modeled according to their electron density. (b) Stereographic representation of three unliganded human Fc regions separately superimposed through their respective CH2 and CH3 domains. Polypeptides are color-coded as follows: blue, 2ql1 (Oganesyan et al., 2008 ▶); red, 1h3w (Krapp et al., 2003 ▶); green, Fc/TM.

Figure 2

Stereographic superimposition of Fc/TM with the CD16-liganded human Fc structure corresponding to PDB code 1e4k (Sondermann et al., 2000 ▶). Superimposition of Cα atoms of Fc/TM and 1e4k (CH2 domain residues 236–342) was carried out using LSQKAB (Kabsch, 1976 ▶). For both chains of 1e4k, the r.m.s. displacement was estimated at 1.9 Å, with a maximum displacement of 6.4 Å for the Cα of residue 286. Human CD16 and Fc/TM are colored green and orange, respectively. Chains A and B of 1e4k are colored red and blue, respectively.

Figure 3

Analysis of the binding of human (a) CD64, (b) CD32A, (c) CD16/V158 and (d) C1q to mAb 3649 and 3649/TM. Measurements were carried out using an ELISA format as described in §2. Errors were estimated as the standard deviations of at least two individual measurements for each interacting pair. The illegibility of the error bars for some data points is the direct result of the corresponding very low standard deviations. Data shown are representative of at least two independent series of experiments.