Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P1−(5'−Adenosyl) P5−(5'−Uridyl) Pentaphosphate (UP5A) and Mg2+ at 2.2 Å: Implications for Water−Mediated Specificity

The three−dimensional structure of the UMP/CMP kinase (UK) from the slime mold Dictyostelium discoideum complexed with the specific and asymmetric bisubstrate inhibitor P1−(5'−adenosyl) P5−(5'−uridyl) pentaphosphate (UP5A) has been determined at a resolution of 2.2 A. The structure of the enzyme, which has up to 41% sequence homology with known adenylate kinases (AK), represents a closed conformation with the flexible monophosphate binding domain (NMP site) being closed over the uridyl moiety of the dinucleotide. Two water molecules were found within hydrogen−bonding distance to the uracil base. The key residue for the positioning and stabilization of those water molecules appears to be asparagine 97, a residue that is highly specific for AK−homologous UMP kinases, but is almost invariably a glutamine in adenylate kinases. Other residues in this region are highly conserved among AK−related NMP kinases. The catalytic Mg2+ ion is coordinated with octahedral geometry to four water molecules and two oxygens of the phosphate chain of UP5A but has no direct interactions with the protein. The comparison of the geometry of the UKdicty.UP5A.Mg2+ complex with the previously reported structure of the UKyeast.ADP.ADP complex [Muller−Dieckmann & Schulz (1994) J. Mol. Biol. 236, 361−367] suggests that UP5A in our structure mimics an ADP.Mg.UDP biproduct inhibitor rather than an ATP. MG.UMP bisubstrate inhibito

Crystal structure of the motor domain of a class-I myosin

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is ∼30° further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain

Structure of a genetically engineered molecular motor

Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 Å resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 Å conformational amplifier consisting of repeats 1 and 2 of α-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosins catalytically active closed conformation. Finally, we describe the structure of -actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted -helix. This fold is very similar to the previously described structures of -actinin repeats 2 and 3, and -spectrin repeats 16 and 17