The pH Dependence of CD2 Domain 1 Self-Association and 15N Chemical Exchange Broadening Is Correlated with the Anomalous pKa of Glu41

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Abstract
We have previously shown using (15)N nuclear relaxation measurements that the concentration-dependent rotational correlation time and chemical exchange broadening for selected resonances of rat CD2 domain 1 (CD2d1) are consistent with a model of low-affinity self-association of the protein molecules. The exchange broadening data, which at high protein concentrations highlight selected nuclei in the major C'-C-F-G beta-sheet face of the immunoglobulin fold, implicate a surface reminiscent of the major lattice contact within crystals of the intact CD2 ectodomain. In a separate study, we have also demonstrated that the beta-strand C' surface-exposed residue Glu41 possesses an anomalously elevated acidity constant (pK(a) = 6.7 at a protein concentration of 1.2 mM). Mutagenesis studies showed that the close contact of residue Glu41 with Glu29 (beta-strand C) is the primary cause of the high pK(a). However, the measured pK(a) of Glu41 also shows a weak dependence on protein concentration, implicating Glu41 in the mechanism of CD2d1 self-association. In the study presented here, we demonstrate a correlation of the pH dependence of the chemical shift and (15)N nuclear relaxation parameters measured for wild-type and mutant forms of CD2d1 with pH and the protonation state of Glu41. Self-association of CD2d1 molecules is promoted whenever the side chain charge of residue 41 is neutralized. These observations are consistent with a model for CD2d1 self-association that corresponds to the crystal structure lattice contact where the interatomic distances are consistent with Glu41 being in the protonated state. This study reinforces the conclusion that residue-specific chemical exchange broadening of protein resonances can arise from weak self-association phenomena. In addition, the electrostatic profile of rat CD2 interfacial residues parallels that of the homologous human CD2 in a manner that suggests a rationalization of similar exchange broadening observations.