Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin.
Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.
Departments of Pathology and Ophthalmology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095, USA.
Oktay K
OK
Gasymov
Adil R
AR
Abduragimov
Taleh N
TN
Yusifov
Ben J
BJ
Glasgow
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D054835:Lipocalin 1; D009154:Mutation / Q000235:genetics; D010169:Palmitic Acids / Q000494:pharmacology; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013666:Tears / Q000737:chemistry*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D005227:Fatty Acids; C518443:LCN1 protein, human; D008024:Ligands; D054835:Lipocalin 1; D010169:Palmitic Acids; C047265:16-(9-anthroyloxy)palmitic acid; D014364:Tryptophan