2PHI: A Large Conformational Change Is Found In The Crystal Structure Of The Porcine Pancreatic Phospholipase A2 Point Mutant F63v

Citation:
Abstract
The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.
PDB ID: 2PHIDownload
MMDB ID: 2859
PDB Deposition Date: 1993/4/8
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 2.2  Å
Source Organism:
Similar Structures:
Biological Unit for 2PHI: dimeric; determined by author and by software (PISA)
Molecular Components in 2PHI
Label Count Molecule
Proteins (2 molecules)
2
Phospholipase A2(Gene symbol: PLA2G1B)
Molecule annotation
Chemicals (3 molecules)
1
3
* Click molecule labels to explore molecular sequence information.

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