Structural Analysis of Phospholipase A₂ from Functional Perspective. 1. Functionally Relevant Solution Structure and Roles of the Hydrogen-Bonding Network^,

Abstract

Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

“…20 In the present work, when the temperature was lowered to 285 K, a cross-peak was clearly detected at 11.46 N when the sample was titrated to pH 5.1 (Figure 7(b)). The proton resonance at 13.02 ppm has been mentioned above (Figure 3 Figure 7(c)), accompanied by the appearance of the most downfield proton resonance at 18.00 ppm.…”

“…The lowest pH, at which the downfield peaks were investigated and observed, was 3.8, and the highest was 4.8. These problems made bee venom sPLA 2 unfavorable for further investigations and prompted us to switch to bovine pancreatic sPLA 2 , whose solution structure has already been solved by NMR at neutral pH 20 and which contains only two histidine residues. The wild-type (WT) bovine pancreatic sPLA 2 has been shown to be stable against pH and chemical denaturation.…”

“…The normal H-bond between the active site Asp and His in sPLA 2 may serve to fix the orientation of the histidine imidazole ring. 5,20 Materials and Methods…”

“…Both NMR studies 20 and the recent 0.97 Å ultrahigh resolution crystal structure 36 of free bovine pancreatic sPLA 2 show that H48 and D99 form a H-bond involving the N 12 of the imidazole ring. Taken together, we conclude that the LBHB is formed between H48 and the inhibitor.…”

Dynamics of Ordered Water in Interfacial Enzyme Recognition: Bovine Pancreatic Phospholipase A2

“…Conformational changes have been observed by NMR spectroscopy. [4,5] In reference [5], it was demonstrated that PLA2 is not denatured by the anionic surfactant sodium dodecyl sulfate (SDS) at a much higher concentration (50 mm) than its critical micellar concentration (% 1 mm)-protein-micelle complexes are formed. The study also indicated that when bound to the micelle, the overall protein structure remains intact, although a more-ordered helical conformation is observed at the Nterminus of the protein.…”