Striatins contain a noncanonical coiled coil that binds protein phosphatase 2A A subunit to form a 2:2 heterotetrameric core of striatin-interacting phosphatase and kinase (STRIPAK) complex.

The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.

X-ray solution scattering of squid heavy meromyosin: strengthening the evidence for an ancient compact off state.

The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the "off" state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the "on" state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca(2+) the radius of gyration increases. Differences in the squid "on" and "off" states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca(2+)-free squid heavy meromyosin that is compact, but which becomes extended when Ca(2+) is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the "off" state is in excellent agreement with the measured "off" state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.

Deriving how far structural information is transmitted through parallel homodimeric coiled-coils: a correlation analysis of helical staggers.

How local conformation is affected by local sequence is fairly well understood for alpha-helical coiled-coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out-of-register alignments, or so-called "staggers" between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two "signs," where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled-coils are commonly influenced by attached structures. Thus, the structures of coiled-coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled-coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled-coils for signed staggers separated by the 40-residue actin repeat distance, consistent with the semi-flexible tropomyosin filament binding F-actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main-chain hydrogen bond deformations.

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.

How sequence directs bending in tropomyosin and other two-stranded alpha-helical coiled coils.

A quantitative analysis of the direction of bending of two-stranded alpha-helical coiled coils in crystal structures has been carried out to help determine how the amino acid sequence of the coiled coil influences its shape and function. Change in the axial staggering of the coiled coil, occurring at the boundaries of either clusters of core alanines in tropomyosin or of clusters of core bulky residues in the myosin rod, causes bending within the plane of the local dimer. The results also reveal that large gaps in the core of the coiled coil, which are seen for small core residues near large core residues or for unbranched core residues near canonical branched core residues, are correlated with bending out of the local dimeric plane. Comparison of tropomyosin structures determined in independent crystal environments provides further evidence for the concept that sequence directs the bending of the coiled coil, but that crystal environment is at least as important as sequence for determining the magnitude of bending. Tropomyosin thus appears to consist of more directionally restrained hinge-like joints rather than directionally variable universal joints, which helps account for and predicts the geometric and dynamic nature of its binding to F-actin.

An unstable head-rod junction may promote folding into the compact off-state conformation of regulated myosins.

The N-terminal region of myosin's rod-like subfragment 2 (S2) joins the two heads of this dimeric molecule and is key to its function. Previously, a crystal structure of this predominantly coiled-coil region was determined for a short fragment (51 residues plus a leucine zipper) of the scallop striated muscle myosin isoform. In that study, the N-terminal 10-14 residues were found to be disordered. We have now determined the structure of the same scallop peptide in three additional crystal environments. In each of two of these structures, improved order has allowed visualization of the entire N-terminus in one chain of the dimeric peptide. We have also compared the melting temperatures of this scallop S2 peptide with those of analogous peptides from three other isoforms. Taken together, these experiments, along with examination of sequences, point to a diminished stability of the N-terminal region of S2 in regulated myosins, compared with those myosins whose regulation is thin filament linked. It seems plain that this isoform-specific instability promotes the off-state conformation of the heads in regulated myosins. We also discuss how myosin isoforms with varied thermal stabilities share the basic capacity to transmit force efficiently in order to produce contraction in their on states.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

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.

Breaking symmetry in protein dimers: designs and functions.

Symmetry, and in particular point group symmetry, is generally the rule for the global arrangement between subunits in homodimeric and other oligomeric proteins. The structures of fragments of tropomyosin and bovine fibrinogen are recently published examples, however, of asymmetric interactions between chemically identical chains. Their departures from strict twofold symmetry are based on simple and generalizable chemical designs, but were not anticipated prior to their structure determinations. The current review aims to improve our understanding of the structural principles and functional consequences of asymmetric interactions in proteins. Here, a survey of >100 diverse homodimers has focused on the structures immediately adjacent to the twofold axis. Five regular frameworks in alpha-helical coiled coils and antiparallel beta-sheets accommodate many of the twofold symmetric axes. On the basis of these frameworks, certain sequence motifs can break symmetry in geometrically defined manners. In antiparallel beta-sheets, these asymmetries include register slips between strands of repeating residues and the adoption of different side-chain rotamers to avoid steric clashes of bulky residues. In parallel coiled coils, an axial stagger between the alpha-helices is produced by clusters of core alanines. Such simple designs lead to a basic understanding of the functions of diverse proteins. These functions include regulation of muscle contraction by tropomyosin, blood clot formation by fibrin, half-of-site reactivity of caspase-9, and adaptive protein recognition in the matrix metalloproteinase MMP9. Moreover, asymmetry between chemically identical subunits, by producing multiple equally stable conformations, leads to unique dynamic and self-assembly properties.

Structure of the mid-region of tropomyosin: bending and binding sites for actin.

Tropomyosin is a two-chain alpha-helical coiled coil whose periodic interactions with the F-actin helix are critical for thin filament stabilization and the regulation of muscle contraction. Here we deduce the mechanical and chemical basis of these interactions from the 2.3-A-resolution crystal structure of the middle three of tropomyosin's seven periods. Geometrically specific bends of the coiled coil, produced by clusters of core alanines, and variable bends about gaps in the core, produced by isolated alanines, occur along the molecule. The crystal packing is notable in signifying that the functionally important fifth period includes an especially favorable protein-binding site, comprising an unusual apolar patch on the surface together with surrounding charged residues. Based on these and other results, we have constructed a specific model of the thin filament, with the N-terminal halves of each period (i.e., the so-called "alpha zones") of tropomyosin axially aligned with subdomain 3 of each monomer in F-actin.

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head.

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.

Visualization of an unstable coiled coil from the scallop myosin rod.

Alpha-helical coiled coils in muscle exemplify simplicity and economy of protein design: small variations in sequence lead to remarkable diversity in cellular functions. Myosin II is the key protein in muscle contraction, and the molecule's two-chain alpha-helical coiled-coil rod region--towards the carboxy terminus of the heavy chain--has unusual structural and dynamic features. The amino-terminal subfragment-2 (S2) domains of the rods can swing out from the thick filament backbone at a hinge in the coiled coil, allowing the two myosin 'heads' and their motor domains to interact with actin and generate tension. Most of the S2 rod appears to be a flexible coiled coil, but studies suggest that the structure at the N-terminal region is unstable, and unwinding or bending of the alpha-helices near the head-rod junction seems necessary for many of myosin's functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-A-resolution crystal structure of a leucine-zipper-stabilized fragment of the scallop striated-muscle myosin rod adjacent to the head-rod junction. The N-terminal 14 residues are poorly ordered; the rest of the S2 segment forms a flexible coiled coil with poorly packed core residues. The unusual absence of interhelical salt bridges here exposes apolar core atoms to solvent.

The crystal structure of the C-terminal fragment of striated-muscle alpha-tropomyosin reveals a key troponin T recognition site.

Contraction in striated and cardiac muscles is regulated by the motions of a Ca(2+)-sensitive tropomyosin/troponin switch. In contrast, troponin is absent in other muscle types and in nonmuscle cells, and actomyosin regulation is myosin-linked. Here we report an unusual crystal structure at 2.7 A of the C-terminal 31 residues of rat striated-muscle alpha-tropomyosin (preceded by a fragment of the GCN4 leucine zipper). The C-terminal 22 residues (263-284) of the structure do not form a two-stranded alpha-helical coiled coil as does the rest of the molecule, but here the alpha-helices splay apart and are stabilized by the formation of a tail-to-tail dimer with a symmetry-related molecule. The site of splaying involves a small group of destabilizing core residues that is present only in striated muscle tropomyosin isoforms. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles.