Ariel Lustig

A distinct 14 residue site triggers coiled‐coil formation in cortexillin I

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two‐stranded, parallel coiled‐coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311–Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled‐coil formation. Moreover, deletion of the six N‐terminal heptad repeats of Ir led to the formation of a four‐ rather than a two‐helix structure, suggesting that the full‐length cortexillin I coiled‐coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled‐coil ‘trigger’ site Arg311–Asp324 yielded ∼30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptides helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311–Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled‐coil formation of cortexillin I.

Dissection of a (betaalpha)8-barrel enzyme into two folded halves.

The (βα)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (βα) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consists of the four N-terminal (βα) units and HisF-C of the four C-terminal (βα) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (βα)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.

TENASCIN-C HEXABRACHION ASSEMBLY IS A SEQUENTIAL TWO-STEP PROCESS INITIATED BY COILED-COIL ALPHA -HELICES

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression inEscherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Op18/stathmin caps a kinked protofilament‐like tubulin tetramer

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended α‐helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C‐terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned α/β‐tubulin heterodimers. The appearance of the complex was that of a kinked protofilament‐like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N‐terminal part of the molecule is to ‘cap’ tubulin subunits to ensure the specificity of the complex and (ii) the complete C‐terminal α‐helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.

Improving Coiled-coil Stability by Optimizing Ionic Interactions

Alpha-helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation. We show that all peptides are highly alpha-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.

The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded α-helical bundle between residues 20 and 83

The N‐terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20–83, was over‐expressed in E. coli and purified under non‐denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly α‐helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five‐stranded α‐helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid‐cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin‐like α‐helical coiled‐coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a ‘doggy‐bone’. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6‐ to 8‐mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two‐dimensional lattices in a time‐dependent manner.

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.

Coiled‐coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve–muscle synapse, binds to the three‐stranded coiled‐coil domain of laminin‐1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin‐1 coiled coil in detail. Binding assays with individual laminin‐1 full‐length chains and fragments revealed that agrin specifically interacts with the γ1 subunit of laminin‐1, whereas no binding to α1 and β1 chains was detected. By using recombinant γ1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled‐coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant γ1 fragments bound at least 10‐fold less than native laminin‐1 indicates that the structure of the three‐stranded coiled‐coil domain of laminin is required for high‐affinity agrin binding. Interestingly, no binding to a chimeric γ2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.

Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements.

Ultrasonic absorption and velocity dispersion curves have been measured in the temperature induced helix-coil transition range of poly-N5-(3-hydroxypropyl)-L-glutamine in a methanol/water mixture. The results clearly reflect an effect due to the kinetics of the conformational conversion. A practically single relaxation time is observed which passes through a maximum when plotted versus the degree of transition. This maximum occurs at definitely less than 50% helix as predicted for by the theory for the comparatively short chain length involved here. The results are discussed in relation to previous theoretical and experimental findings.

Dimerization Regulates the Enzymatic Activity of Escherichia coli Outer Membrane Phospholipase A

The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca2+ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.