Crystal structure of a high-affinity variant of rat alpha-parvalbumin.
In model peptide systems, Ca2+ affinity is maximized in EF-hand motifs containing four carboxylates positioned on the +x and -x and +z and -z axes; introduction of a fifth carboxylate ligand reduces the affinity. However, in rat beta-parvalbumin, replacement of Ser-55 with aspartate heightens divalent ion affinity [Henzl, M. T., et al. (1996) Biochemistry 35, 5856-5869]. The corresponding alpha-parvalbumin variant (S55D/E59D) likewise exhibits elevated affinity [Henzl, M. T., et al. (2003) Anal. Biochem. 319, 216-233]. To determine whether these mutations produce a variation on the archetypal EF-hand coordination scheme, we have obtained high-resolution X-ray crystallographic data for alpha S55D/E59D. As anticipated, the aspartyl carboxylate replaces the serine hydroxyl at the +z coordination position. Interestingly, the Asp-59 carboxylate abandons the role it plays as an outer sphere ligand in wild-type rat beta, rotating away from the Ca2+ and, instead, forming a hydrogen bond with the amide of Glu-62. Superficially, the coordination sphere in the CD site of alpha S55D/E59D resembles that in the EF site. However, the orientation of the Asp-59 side chain is predicted to stabilize the D-helix, which may contribute to the heightened divalent ion affinity. DSC data indicate that the alpha S55D/E59D variant retains the capacity to bind 1 equiv of Na+. Consistent with this finding, when binding measurements are conducted in K(+)-containing buffer, divalent ion affinity is markedly higher. In 0.15 M KCl and 0.025 M Hepes-KOH (pH 7.4) at 5 degrees C, the macroscopic Ca2+ binding constants are 1.8 x 10(10) and 2.0 x 10(9) M(-1). The corresponding Mg2+ binding constants are 2.7 x 10(6) and 1.2 x 10(5) M(-1).
Department of Chemistry, University of Missouri, Columbia, Missouri 65211, USA.
Yong-Hwan
YH
Lee
John J
JJ
Tanner
John D
JD
Larson
Michael T
MT
Henzl
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D002413:Cations, Divalent; D003460:Crystallization; D018360:Crystallography, X-Ray; D020832:EF Hand Motifs; D018698:Glutamic Acid / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D051381:Rats; D012694:Serine / Q000235:genetics; D012996:Solutions
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D010320:Parvalbumins; D020033:Protein Isoforms; D012996:Solutions; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D008274:Magnesium; D011188:Potassium; D002118:Calcium