Jerry H. Brown

Heptad breaks in α‐helical coiled coils: Stutters and stammers

The discontinuities found in heptad repeats of α‐helical coiled‐coil proteins have been characterized. A survey of 40 α‐fibrous proteins reveals that only two classes of heptad breaks are prevalent: the stutter, corresponding to a deletion of three residues, and the newly identified “stammer,” corresponding to a deletion of four residues. This restriction on the variety of insertions/deletions encountered gives support to a unifying structural model, where different degrees of supercoiling accommodate the observed breaks. Stutters in the hemagglutinin coiled‐coil region have previously been shown to produce an underwinding of the supercoil, and we show here how, in other cases, stammers would lead to overwinding. An analysis of main‐chain structure also indicates that the mannose‐binding protein, as well as hemagglutinin, contains an underwound coiled‐coil region. In contrast to knobs‐into‐holes packing, these models give rise to non‐close‐packed cores at the sites of the heptad phase shifts. We suggest that such non‐close‐packed cores may function to terminate certain coiled‐coil regions, and may also account for the flexibility observed in such long α‐fibrous molecules as myosin. The local underwinding or overwinding caused by these specific breaks in the heptad repeat has a global effect on the structure and can modify both the assembly of the protein and its interaction properties.

Deciphering the design of the tropomyosin molecule

The crystal structure at 2.0-Å resolution of an 81-residue N-terminal fragment of muscle α-tropomyosin reveals a parallel two-stranded α-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (≈1.2-Å) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded α-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

Regulation of Muscle Contraction by Tropomyosin and Troponin: How Structure Illuminates Function

Publisher Summary This chapter provides a brief description of tropomyosins periodic and aperiodic structural features related to their function. The chapter describes the structure of troponin and provides information on F-actin. The thin filaments of vertebrate striated muscle are periodic structures composed of three proteins with different designs that function together for the regulation of contraction. Tropomyosin displays two types of periodicity. In addition to a long-range ∼40-residue repeat, tropomyosin is the paradigm of the α-helical coiled-coil class of proteins. The seven distinct amino acid positions and associated interactions that are produced from the α-helical coiled-coil provide the basic structural unit of tropomyosin. These elements superimpose on the longer, roughly 40-residue, functional unit of tropomyosin and patterns of residues found both in the core of the coiled-coil and on its surface are repeated seven times in a full-length tropomyosin molecule; and they play a role in the periodic binding of tropomyosin to actin.

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

Contraction in striated and cardiac muscles is regulated by the motions of a Ca2+-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 Å of the C-terminal 31 residues of rat striated-muscle α-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 α-helical coiled coil as does the rest of the molecule, but here the α-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.

Visualization of an unstable coiled coil from the scallop myosin rod

α-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 molecules two-chain α-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 α-helices near the head–rod junction seems necessary for many of myosins functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-Å-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.

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 α‐helical coiled coils and antiparallel β‐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 β‐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 α‐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.

The structure of a membrane fusion mutant of the influenza virus haemagglutinin

The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431‐439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp‐‐‐‐Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra‐chain hydrogen bonds that stabilize the location of the N‐terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N‐terminus at higher pH.

Crystal structure of the central region of bovine fibrinogen (E5 fragment) at 1.4-Å resolution

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E5 fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular “handshake” by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the Aα and Bβ chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal γ chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two γ chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E5 fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin.

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

Background: Striatins are novel regulatory subunits of PP2A in the striatin-interacting phosphatase and kinase (STRIPAK) complex. Results: The striatin coiled coil forms a noncanonical dimer required for PP2A A subunit binding. Conclusion: The coiled coil of striatins bind PP2A A subunits to form a 2:2 heterotetrameric core of the STRIPAK complex. Significance: The current structural analysis provides insights into the assembly of the 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.

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.