Purification, crystallization and preliminary X-ray crystallographic analysis of squid heavy meromyosin.

All muscle-based movement is dependent upon carefully choreographed interactions between the two major muscle components, myosin and actin. Regulation of vertebrate smooth and molluscan muscle contraction is myosin based (both are in the myosin II class), and requires the double-headed form of myosin. Removal of Ca2+ from these muscles promotes a relatively compact conformation of the myosin dimer, which inhibits its interaction with actin. Although atomic structures of single myosin heads are available, the structure of any double-headed portion of myosin, including the ∼375 kDa heavy meromyosin (HMM), has only been visualized at low (∼20 Å) resolution by electron microscopy. Here, the growth of three-dimensional crystals of HMM with near-atomic resolution (up to ∼5 Å) and their X-ray diffraction are reported for the first time. These crystals were grown in off-state conditions, that is in the absence of Ca2+ and the presence of nucleotide analogs, using HMM from the funnel retractor muscle of squid. In addition to the crystallization conditions, the techniques used to isolate and purify this HMM are also described. Efforts at phasing and improving the resolution of the data in order to determine the structure are ongoing.

Crystal structure of a phosphorylated light chain domain of scallop smooth-muscle myosin.

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg(16), an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin.

We have determined the 2.3-Å-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ("smooth") muscle. This structure reveals hinges that may function in the "on" and "off" states of myosin. The molecule adopts two different conformations about the heavy chain "hook" and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 Å. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

Crystallization and preliminary X-ray diffraction studies of NusG, a protein shared by the transcription and translation machines.

N-utilization factor G (NusG) from Aquifex aeolicus (Aa) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion technique. The drops consisted of 2.5 microl protein solution (approximately 30 mg ml(-1) in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM EDTA and 10 mM DTT) and 2.5 microl reservoir solution (0.085 M Na HEPES pH 7.5, 15% glycerol, 11% 2-propanol and 20% PEG 4000) derived from condition number 41 of the Hampton Cryo Screen. The crystals grew at 291 +/- 1 K and reached dimensions of 0.2 x 0.1 x 0.05 mm in 5-7 d. The crystals, which diffracted to 2.45 A resolution, belonged to space group C222(1), with unit-cell parameters a = 65.95, b = 124.58, c = 83.60 A. One AaNusG molecule is present in the asymmetric unit, corresponding to a solvent content of 59.80% (Matthews coefficient = 3.06 A(3) Da(-1)). Crystal structure determination is in progress.

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.