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 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-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 myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.

Stepwise modulation of ATPase activity, nucleotide trapping, and sliding motility of myosin S1 by modification of the thiol region with residues of increasing size.

Rabbit muscle myosin S1 was modified either at SH1 alone or at both SH1 and SH2, using a series of alkylthiolating reagents of increasing size, designed for correlating gradually changing structural disturbances in the thiol region with functional impairments in the myosin head. The reagents were of the type H(CH(2))(n)()-S-NTB, (NTB = 2-nitro-5-thiobenzoate) (n = 1, 2, 5, 8, 9, 10, 11, and 12). Modification of only SH1 led to the expected activation of the Ca(2+)-ATPase, but only with small reagents, while reagents with n > or = 10 caused inhibition of the Ca(2+)-ATPase. Modification of both SH1 and SH2 showed the expected inhibition of Ca(2+)-ATPase but likewise allowed considerable residual Ca(2+)-ATPase activity if the residues were small. Trapping of the nucleotide, known to occur with cross-linking reagents, was seen also with monovalent reagents, provided their length exceeded n = 9 or 10. All S1 derivatives prepared in this study possessed an affinity for actin comparable to native S1 but lacked sliding motility in in vitro motility assays. The biochemical data of this study can be related to existing models of myosin S1 and recent structural data [Houdusse, A., Kalabokis, V. N., Himmel, D., Szent-Györgyi, A. G., and Cohen, C. (1999) Cell 97, 459-470] by making the assumptions that modification at SH1 prevents the formation of the SH1 helix mandatory for the transmission of conformational energy and that mobility of the thiol region is a prerequisite for ATPase activity. Immobilization of the thiol region by residues of increasing size apparently leads to lower enzyme activity and, finally, to inhibition of nucleotide exchange.

Thiol-specific cross-linkers of variable length reveal a similar separation of SH1 and SH2 in myosin subfragment 1 in the presence and absence of MgADP.

A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].

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 A: 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 [Müller-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 inhibitor.

Crystallization and preliminary X-ray analysis of UMP/CMP-kinase from Dictyostelium discoideum with the specific bisubstrate inhibitor P1-(adenosine 5')-P5-(uridine 5')-pentaphosphate (UP5A).

UMP/CMP-kinase (UK) from the slime mold Dictyostelium discoideum has been purified to high homogeneity and co-crystallized with the bisubstrate inhibitor P1-(adenosine 5')-P5-(uridine 5')-pentaphosphate (UP5A). UP5A binds to UK with a dissociation constant (Kd) of 3 +/- 0.5 nM at 25 degrees C and pH 7.5. This is some 50-fold tighter than the binding of P1,P5-(diadenosine 5')-pentaphosphate (AP5A, Kd = 160 +/- 15 nM). AP5A is a bisubstrate inhibitor that is specific for adenylate kinase. The crystals have the symmetry of the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2. The unit cell dimensions are a = b = 78.5 A and c = 101.4 A. The crystals diffract to a Bragg spacing of 2.1 A.