Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

Here we report a 2.3-Å crystal structure of scallop myosin S1 complexed with ADP⅐BeF x, as well as three additional structures (at 2.8 -3.8 Å resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-Å scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter͞lever arm module from the motor and allows crosslinking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-Å nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release.T he myosin head (subfragment one, or S1) consists of a motor domain (MD) and a lever arm that amplifies small conformational changes of the motor (Fig. 1). The MD comprises four subdomains (the 50-kDa upper and lower subdomains, the N-terminal subdomain, and the converter) linked by three flexible single-stranded joints (switch II, the relay, and the so-called SH1 helix). Motor function results from coupled rigid body rearrangements of the subdomains coordinated with conformational changes in the joints (1, 2). During the acto-myosin contractile cycle (3-5), the MD undergoes a number of conformational changes as the myosin head transduces ATP hydrolysis into mechanical work. The affinity of myosin for actin in the cycle is often described in terms of ''strong'' and ''weak'' actin-binding states. Myosin crystal structures have thus far been obtained only in the absence of actin, and three conformations have been identified, but their place in the contractile cycle has been controversial (6). The first or near-rigor conformation (7), was obtained in the absence of nucleotide, and, because the lever arm orientation was similar to that observed in electron microscopy (EM) images of actomyosin in the rigor state, this conformation was thought to be similar to a true rigor (i.e., strong actin-binding, nucleotide-free) state. More recent studies (8, 9) have shown, however, that this conformation corresponds to a weak actin-binding state occurring shortly after myosin detaches from actin, and that this state can bind to but cannot hydrolyze ATP. I...

X-ray diffraction data at atomic resolution to 0.98 A with 136 380 observed unique reflections were collected using a high quality proteinase K crystals grown under microgravity conditions and cryocooled. The structure has been refined anisotropically with REFMAC and SHELX-97 with R-factors of 11.4 and 12.8%, and R(free)-factors of 12.4 and 13.5%, respectively. The refined model coordinates have an overall rms shifts of 0.23 A relative to the same structure determined at room temperature at 1.5 A resolution. Several regions of the main chain and the side chains, which were not observed earlier have been seen more clearly. For example, amino acid 207, which was reported earlier as Ser has been clearly identified as Asp. Furthermore, side-chain disorders of 8 of 279 residues in the polypeptide have been identified. Hydrogen atoms appear as significant peaks in the F(o) - F(c) difference electron density map accounting for an estimated 46% of all hydrogen atoms at 2sigma level. Furthermore, the carbon, nitrogen, and oxygen atoms can be differentiated clearly in the electron density maps. Hydrogen bonds are clearly identified in the serine protease catalytic triad (Ser-His-Asp). Furthermore, electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. Though unusual, these features seem to be conserved in other serine proteases. Finally there are clear electron density peaks for the hydrogen atoms associated with the Ogamma of Ser 224 and Ndelta1 of His 69.

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.

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Sodium dodecyl sulphate (SDS), an anionic surfactant that mimics some characteristics of biological membrane has also been found to induce aggregation in proteins. The present study was carried out on 25 diverse proteins using circular dichroism, fluorescence spectroscopy, dye binding assay and electron microscopy. It was found that an appropriate molar ratio of protein to SDS readily induced amyloid formation in all proteins at a pH below two units of their respective isoelectric points (pI) while no aggregation was observed at a pH above two units of pI. We also observed that electrostatic interactions play a leading role in the induction of amyloid. This study can be used to design or hypothesize a molecule or drug, which may counter act the factor responsible for amyloid formation.

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å , in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica.In bacteria and plants, L-cysteine is synthesized from L-Serine by two key enzymes serine acetyltransferase (SAT) 3 and O-acetyl serine sulfhydrylase (OASS). SAT converts L-Serine to O-acetyl serine by transferring an acetyl group from acetylCoA. Later, OASS converts the O-acetyl serine (OAS) to L-cys-

Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-Å x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm Ϸ45°to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called ''front door.'' In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-Å nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.M yosin is a motor protein that transduces ATP hydrolysis into mechanical work, leading to its translocation along filamentous actin. It is believed that the small conformational changes induced by enzymatic activity in the myosin ''motor domain'' (MD) are amplified by the motion of the ''lever arm.'' Three weak actin-binding (actin-detached) states have been identified crystallographically for scallop myosin subfragment 1 (S1) (1). These and other structures reveal that the motor function of myosin S1 is coordinated by three subdomains (50-kDa upper and lower subdomains and the converter) that rotate as rigid bodies around the relatively stable N-terminal subdomain (1-3). These rotations depend on conformational changes in three flexible joints (switch II, relay, and the SH1 helix) between the subdomains. Three-dimensional reconstructions of electron microscopic images of actin decorated by S1 of several myosin isoforms (4-6) have led to the suggestion that actin binds to portions of the 50-kDa upper and lower subdomains, whereas various crystal structures (see, for example, refs. 3 and 7-9) indicate that ATP binds at the opposite side of the myosin head in a pocket between the 50-kDa upper and Nterminal subdomains (Figs. 1 and 2A).One of the three structural conformations, the so-called ''prepower stroke,'' corresponds biochemically to the ADP⅐P i transition state (1,8,(10)(11)(12). Here, S1 displays a primed lever arm (Ϸ90°to the actin filament axis), closure of the 50-kDa cleft's base (which is near the nucleotide-binding pocket), and a bent switch II that interacts with both the nucleotide and switch I (a loop in the 50-kDa upper subdomain that coordinates the nucleotide). After this state, the tight binding of myosin to actin results in the so-called ''power stroke'' (after which the lever arm is Ϸ45°to the actin filament), a process believed to be initiated by the near-complete closing of the 50-kDa cleft (4-6). This...