The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody

The Fv fragment from an anti-dansyl antibody was optimally crystallized int o two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high r esolution at 112 K (at 1.45 Angstrom for the crystal at PH 5.25 and at 1.55 Angstrom for that at pH 6.75). In the two crystal structures, marked diffe rences were identified in the first half of CDRH3 s having an amino acid se quence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro100-Trp100aH-Phe100bH-Ala10 1H-Tyr102H. NMR pH titration experiments revealed the pK(a) values of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (pK(a) = 6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structu res including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His 93L and His55H were almost invariant between the two crystal structures, th ose around His98Hs showed great difference in spite of the small conformati onal difference of His98H between the two crystal structures. These spectro scopic and crystallographic findings suggested that the change in the proto nation state in His98H was responsible for the structural differences betwe en PH 5.25 and 6.75. In addition, the most plausible binding site of the da nsyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 an d the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue f or the binding mode of the dansyl group in the site. (C) 1999 Academic Pres s.