X-ray Structure of the Magnesium(II)·ADP·Vanadate Complex of the Dictyostelium discoideum Myosin Motor Domain to 1.9 Å Resolution

• Published in: Biochemistry (Easton) 1996-04, Vol.35 (17), p.5404-5417
• Main Authors: Smith, Clyde A, Rayment, Ivan
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi952633+

The structure of the vanadate-trapped ADP complex of a truncated head of Dictyostelium myosin II consisting of residues Asp 2−Asn 762 has been determined by molecular replacement at 1.9 Å resolution and refined to a crystallographic R-factor of 19.4%. The crystals belong to the orthorhombic space group C2221 where a = 84.50 Å, b = 145.4 Å, and c = 152.8 Å. The conformation of the protein is similar to that of MgADP·AlF4·S1Dc [Fisher, A. J., et al. (1995) Biochemistry 34, 8960−8972]. The nucleotide binding site contains a complex between MgADP and vanadate where MgADP exhibits a very similar conformation to that seen in previous complexes. The vanadate ion adopts a trigonal bipyramidal coordination. The three equatorial oxygen ligands are fairly short, average 1.7 Å, relative to a single bond distance of ∼1.8 Å and are coordinated to the magnesium ion, Nζ of Lys 185, and five other protein ligands. The apical coordination to the vanadate ion is filled by a terminal oxygen on the β-phosphate of ADP and a water molecule at bond distances of 2.1 and 2.3 Å, respectively. The long length of the apical bonds suggests that the bond order is considerably less than unity. This structure confirms the earlier suggestion that vanadate is a model for the transition state of ATP hydrolysis and thus provides insight into those factors that are responsible for catalysis. In particular, it shows that the protein ligands and water structure surrounding the γ-phosphate pocket are oriented to stabilize a water molecule in an appropriate position for in-line nucleophilic attack on the γ-phosphorus of ATP. This structure reveals also an orientation of the COOH-terminal region beyond Thr 688 which is very different from that observed in either MgADP·BeF x ·S1Dc or chicken skeletal myosin subfragment 1. This is consistent with the COOH-terminal region of the molecule playing an important role in the transduction of chemical energy of hydrolysis of ATP into mechanical movement.