John S. Allingham

The structural basis of blebbistatin inhibition and specificity for myosin II.

Molecular motors play a central role in cytoskeletal-mediated cellular processes and thus present an excellent target for cellular control by pharmacological agents. Yet very few such compounds have been found. We report here the structure of blebbistatin, which inhibits specific myosin isoforms, bound to the motor domain of Dictyostelium discoideum myosin II. This reveals the structural basis for its specificity and provides insight into the development of new agents.

Actin-targeting natural products: structures, properties and mechanisms of action

Abstract.Natural small-molecule inhibitors of actin cytoskeleton dynamics have long been recognized as valuable molecular probes for dissecting complex mechanisms of cellular function. More recently, their potential use as chemotherapeutic drugs has become a focus of scientific investigation. The primary focus of this review is the molecular mechanism by which different actin-targeting natural products function, with an emphasis on structural considerations of toxins for which high-resolution structural information of their interaction with actin is available. By comparing the molecular interactions made by different toxin families with actin, the structural themes of those that alter filament dynamics in similar ways can be understood. This provides a framework for novel synthetic-compound designs with tailored functional properties that could be applied in both research and clinical settings.

Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin

Marine macrolide toxins of trisoxazole family target actin with high affinity and specificity and have promising pharmacological properties. We present X-ray structures of actin in complex with two members of this family, kabiramide C and jaspisamide A, at a resolution of 1.45 and 1.6 Å, respectively. The structures reveal the absolute stereochemistry of these toxins and demonstrate that their trisoxazole ring interacts with actin subdomain 1 while the aliphatic side chain is inserted into the hydrophobic cavity between actin subdomains 1 and 3. The binding site is essentially the same as the one occupied by the actin-capping domain of the gelsolin superfamily of proteins. The structural evidence suggests that actin filament severing and capping by these toxins is also analogous to that of gelsolin. Consequently, these macrolides may be viewed as small molecule biomimetics of an entire class of actin-binding proteins.

An antifreeze protein folds with an interior network of more than 400 semi-clathrate waters.

The crystal structure of an antifreeze protein shows a polypentagonal network of water in the protein core. [Also see Perspective by Sharp] When polypeptide chains fold into a protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich antifreeze protein that retains ~400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the protein interior. The ordered waters extend outwards to the protein surface and likely are involved in ice binding. This protein fold supports both the anchored-clathrate water mechanism of antifreeze protein adsorption to ice and the water-expulsion mechanism of protein folding. Folding When Wet Most globular proteins release water as they fold to form a dry hydrophobic core. In contrast, Sun et al. (p. 795; see the Perspective by Sharp) report a high-resolution structure showing that the antifreeze protein Maxi retains about 400 water molecules in its core. Maxi is a dimer in which two helical monomers each bend in the middle to form a four-helix bundle. The helices are spaced slightly apart to accommodate two intersecting polypentagonal monolayers of water. The pentagons form cages around inward pointing side chains to stabilize the structure. The ordered waters extend to the protein surface where they are likely to be involved in ice binding.

Vik1 Modulates Microtubule-Kar3 Interactions through a Motor Domain that Lacks an Active Site

Conventional kinesin and class V and VI myosins coordinate the mechanochemical cycles of their motor domains for processive movement of cargo along microtubules or actin filaments. It is widely accepted that this coordination is achieved by allosteric communication or mechanical strain between the motor domains, which controls the nucleotide state and interaction with microtubules or actin. However, questions remain about the interplay between the strain and the nucleotide state. We present an analysis of Saccharomyces cerevisiae Kar3/Vik1, a heterodimeric C-terminal Kinesin-14 containing catalytic Kar3 and the nonmotor protein Vik1. The X-ray crystal structure of Vik1 exhibits a similar fold to the kinesin and myosin catalytic head, but lacks an ATP binding site. Vik1 binds more tightly to microtubules than Kar3 and facilitates cooperative microtubule decoration by Kar3/Vik1 heterodimers, and yet allows motility. These results demand communication between Vik1 and Kar3 via a mechanism that coordinates their interactions with microtubules.

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin–spin interactions, as measured by continuous wave EPR for distances of 0.7–2 nm or pulsed EPR (double electron–electron resonance) for distances of 1.7–6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

Biochemical Characterization of UDP-Gal:GlcNAc-Pyrophosphate-Lipid β-1,4-Galactosyltransferase WfeD, a New Enzyme from Shigella boydii Type 14 That Catalyzes the Second Step in O-Antigen Repeating-Unit Synthesis

The O antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and is essential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causes diarrhea in humans. The O antigen of Shigella boydii type 14 consists of repeating oligosaccharide units with the structure [→6-d-Galpα1→4-d-GlcpAβ1→6-d-Galpβ1→4-d-Galpβ1→4-d-GlcpNAcβ1→]n. The wfeD gene in the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a β4-GalT that synthesizes the Galβ1-4GlcNAcα-R linkage. WfeD was expressed in Escherichia coli, and the activity was characterized by using UDP-[³H]Gal as the donor substrate as well as the synthetic acceptor substrate GlcNAcα-pyrophosphate-(CH₂)₁₁-O-phenyl. The enzyme product was analyzed by liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn²(+), Ni²(+), and, surprisingly, also Pb²(+) enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in the binding of the negatively charged acceptor substrate. Our study revealed that the β4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian β4-GalT, although it catalyzes a similar reaction.

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand.

Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain‐1 and calpain‐2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C‐terminal penta‐EF hand (PEF) domains. Calpain‐3, or p94, is a skeletal muscle‐specific isoform that is genetically linked to limb‐girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain‐3 support the suggestion that full‐length calpain‐3 exists as a homodimer. Here, we report the crystallization of calpain‐3′s PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain‐3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain‐3 PEF domain contains a Ca2+ bound at the EF5‐hand used for homodimer association. Three of the four Ca2+‐binding EF‐hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain‐3 large subunit were to try to pair with a calpain small subunit.

Role of Ca2+ in folding the tandem β‐sandwich extender domains of a bacterial ice‐binding adhesin

A Ca2+‐dependent 1.5‐MDa antifreeze protein present in an Antarctic Gram‐negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice‐binding adhesin. The non‐ice‐binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104‐residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104‐residue RII construct (RII monomer). CD demonstrated that Ca2+ is required for RII monomer folding, and that the monomer is fully structured at a Ca2+/protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single‐wavelength anomalous dispersion and molecular replacement methods with Ca2+ as the heavy atom to obtain phase information. The RII monomer folds as a Ca2+‐bound immunoglobulin‐like β‐sandwich. Ca2+ ions are coordinated at the interfaces between each RII monomer and its symmetry‐related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca2+ ions help to rigidify the chain of 104‐residue repeats in order to project the ice‐binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats‐in‐toxin family that have been characterized as adhesins exported via the type I secretion pathway.

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice

Structure of a bacterial adhesin reveals its role in forming a mixed-species symbiotic community with diatoms on sea ice. Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a “dissect and build” structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several “repeats-in-toxin” motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.