Proton nuclear magnetic resonance study on the dynamics of the conformation of the hinge segment of human G1 immunoglobulin

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Abstract
A proton nuclear magnetic resonance (NMR) study is reported for the dynamics of the conformation of the hinge segment of human G1 immunoglobulin. The hinge fragment (Thr222-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu234)2 was obtained by tryptic digestion of F(ab'')2, a peptic fragment of IgG1. Comparisons of the NMR results obtained for the hinge fragment with those for the intact IgG1 and its fragments led us to conclude that (1) a significant change in conformation of the segment preceding the disulfide-linked Cys-Pro-Pro-Cys core is induced when the Fab portion is cleaved off and (2) the presence or absence of the Fc portion affects very little, if any, of the conformation of this part of the hinge. On the basis of the present NMR results along with those which we have obtained previously using the intact IgG1 and its fragments, it was concluded that the conformation of the segment preceding the Cys-Pro-Pro-Cys core of the intact IgG1 can be maintained only when it is flanked by the Fab portion and the Cys-Pro-Pro-Cys core. An X-ray crystallographic study [Marquart, M., Deisenhofer, J., Huber, R., and Palm, W. (1980) J. Mol. Biol. 141, 369-392] showed that segment Cys-220-Thr-225 forms a one-turn helix with little inherent stability. Upon loss of Fab or Fc, residual segments of the hinge would become too short to form the helix. In view of the NMR results obtained for Fab, Fc(t), and the hinge fragment, we suggest that the helical structure of the hinge as revealed by the X-ray crystallographic study is also retained in solution. We have previously shown on the basis of experiments using spin diffusion that the Lys-222-Thr-225 segment of the hinge is exposed to solvent and is primarily responsible for the internal flexibility of the IgG1 molecule. We suggest that the helical structure makes a major contribution to the expression of the flexibility of the IgG1 molecule. Spin coupling patterns for the .alpha. and .beta. protons of Cys-226 and Cys-229 of hinge fragment I were analyzed under a variety of conditions of pH and temperature. It was concluded that the conformation about the C.alpha. and C.beta. bond in Cys-226 and Cys-229 of IgG1 in solution changes very little, if any, throughout the conditions of pH and temperature examined and is quite similar to that in the crystal. Results of NMR measurements along with those obtained by using without sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on an HPLC column show that (1) the segment, which follows the Cys-Pro-Pro-Cys core, is extended and (2) the presence of the disulfide-linked core is essential in maintaining the extended conformation. It was shown that the .beta.-proton chemical shifts for Cys-229 change to a great extent on increasing the temperature, whereas very little shift was observed for Cys-226. Model building shows that there exists a significant degree of freedom of internal motion involving NH-C.alpha. and C.beta.-S bonds of Cys-229 with the dihedral angle around the C.alpha. and C.beta. bond held fixed. We suggest that the internal motion is responsible for the failure of observing the electron density beyond the Cys-Pro-Pro-Cys core in the X-ray crystallographic analyses of IgG1. We also suggest that the internal motion is conjunction with the extended conformation of the segment that follows the Cys-Pro-Pro-Cys core plays an important role in regulating the quaternary structure of the CH2 domains of the optimum C1 binding.