The pH dependence of CD2 domain 1 self-association and 15N chemical exchange broadening is correlated with the anomalous pKa of Glu41.
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.
Chen
H A
HA
Department of Biochemistry and Molecular Biology, University College London, Gower Street, United Kingdom.
Pfuhl
Mark
M
Driscoll
Paul C
PC
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D018801:CD2 Antigens / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes; D002448:Cell Adhesion; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D013816:Thermodynamics
D018801:CD2 Antigens; D002247:Carbon Isotopes; C465878:Cd2 protein, rat; D009587:Nitrogen Isotopes; D005973:Glutamine; D018698:Glutamic Acid