Structure-function analysis of the heat shock factor-binding protein reveals a protein composed solely of a highly conserved and dynamic coiled-coil trimerization domain.

Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait. Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins. Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif. Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high alpha-helical content and moderate stability. NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases. Unlike previously characterized coiled-coils, backbone 15N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region. Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range. NMR analyses suggest a rotationally symmetric trimer. Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.     

Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.     

Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA

Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single alpha-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a beta-sheet subdomain and an alpha-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The beta-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP.     

The carboxy-terminal domain initiates trimerization of bacteriophage T4 fibritin.

Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. Earlier we showed that the C-terminal globular domain (foldon) of fibritin is essential for correct trimerization and folding of the protein. We constructed the chimerical fusion protein W31 in which the fibritin foldon sequence is followed by the small globular non-alpha-helical protein gp31 of the T4 phage. We showed that the foldon is capable of trimerization in the absence of the coiled-coil part of fibritin. A deletion mutant of fibritin (NB1) with completely deleted foldon is unable to fold and trimerize correctly. An excess of this mutant protein did not influence the refolding of fibritin in vitro, and the chimerical protein inhibited this process efficiently. Our conclusion is that the trimerization of the foldon is the initial step of fibritin refolding and is followed by the formation of the coiled-coil structure.     

NMR solution structure of the RED subdomain of the Sleeping Beauty transposase

DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi‐domain protein containing the catalytic and the DNA‐binding domains. The DNA‐binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.     

Crystal structure of the C-terminal 10-kDa subdomain of Hsc70

The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions. They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain. The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain. Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 A. Its helical region adopted a helix-loop-helix fold. This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70. Moreover, in the crystalline state, the 10-kDa subdomain formed dimers. The results of gel filtration chromatography further supported the view that this subdomain was self-associated. Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers. These findings suggest that the alpha-helical region of the 10-kDa subdomain dictates the chaperone self-association.     

Human and Xenopus cingulin share a modular organization of the coiled-coil rod domain: predictions for intra- and intermolecular assembly

The complete nucleotide and derived amino acid sequences of Homo sapiens cingulin cDNA (5143 bp) were determined by sequencing two distinct EST clones that showed significant sequence homology to Xenopus laevis cingulin. Protein sequence analysis indicates that the molecule contains two chains and has a tripartite structure with N-terminal (head) domains, a coiled-coil rod domain (length, 120 nm), and short C-terminal (tail) domains. Human and Xenopus cingulin heads are only 33% identical, yet a human cingulin N-terminal fragment still interacts with canine ZO-1 and ZO-2 in vitro. The rod domain contains two A and two B subdomains, though it lacks the third B subdomain present in Xenopus cingulin. The heptad substructures of Xenopus and human cingulins were further characterized by computer analysis and indicated that the two-stranded coiled-coil structure contained chains that were parallel and in axial register. Fast Fourier transform analysis and a scoring technique designed to recognize potential interactions between different supramolecular arrangements suggests that cingulin dimers may further assemble through antiparallel interactions between the last approximately 100 amino acids of the coiled-coil region. Cingulin mRNA ( approximately 5.2 kb) was detected by Northern blotting in epithelial tissues. A human cingulin EST was mapped to chromosome 1q21 using the UniGene database.     

Tetramerization of human guanylate-binding protein 1 is mediated by coiled-coil formation of the C-terminal α-helices

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 μM. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.     

Spectroscopic studies of fibrinogen and its plasmin-derived fragments.

The secondary structure of human fibrinogen and its plasmin-fragments have been studied by FTIR spectroscopy. The quantitative results for fibrinogen are in good agreement with previous studies using circular dichroism spectroscopy. After treatment of fibrinogen with plasmin in buffer containing Ca2+, two major fragments are produced: fragment E (Mw 45,000) and fragment D (Mw 100,000). Fragment E is shown to contain 50% alpha-helical values, attributed to its coiled-coil portions, and minor beta-strands and turn structures. Its deuteration gives evidence of the presence of solvent-exposed alpha-helical structures. On the other hand, fragment D contains a distribution of secondary structure values of 35% alpha-helix, 29% beta-sheet segments and 17% turn structures. Fragment D itself has two domains: a portion of the original coiled-coil and also a thermally labile globular domain. The coiled-coil portion (Mw 27,000) was isolated and showed a high alpha-helical content (around 70%). The globular domain is estimated to be rich in beta-sheet structures. The spectra of fibrin clots formed in Ca(2+)-containing buffer have a lower amide I/amide II ratio than fibrinogen spectra, which is interpreted as being due to aggregation.     

Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC

Avian reovirus fibre, a homo-trimer of the sigmaC protein, is responsible for primary host cell attachment. The protein expressed in bacteria forms elongated fibres comprised of a carboxy-terminal globular head domain and a slender shaft, and partial proteolysis yielded a carboxy-terminal protease-stable domain that was amenable to crystallisation. Here, we show that this fragment retains receptor-binding capability and report its structure, solved using two-wavelength anomalous diffraction and refined using data collected from three different crystal forms at 2.1 angstroms, 2.35 angstroms and 3.0 angstroms resolution. The carboxy-terminal globular domain has a beta-barrel fold with the same overall topology as the mammalian reovirus fibre (sigma1). However, the monomers of the sigmaC trimer show a more splayed-out arrangement than in the sigma1 structure. Also resolved are two triple beta-spiral repeats of the shaft or stalk domain. The presence in the sequence of heptad repeats amino-terminal to these triple beta-spiral repeats suggests that the unresolved portion of the shaft domain contains a triple alpha-helical coiled-coil structure. Implications for the function and stability of the sigmaC protein are discussed.     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

NMR structure of an F-actin-binding "headpiece" motif from villin

A growing family of F-actin-bundling proteins harbors a modular F-actin-binding headpiece domain at the C terminus. Headpiece provides one of the two F-actin-binding sites essential for filament bundling. Here, we report the first structure of a functional headpiece domain. The NMR structure of chicken villin headpiece (HP67) reveals two subdomains that share a tightly packed hydrophobic core. The N-terminal subdomain contains bends, turns, and a four-residue alpha-helix as well as a buried histidine residue that imparts a pH-dependent folding. The C-terminal subdomain is composed of three alpha-helices and its folding is pH-independent. Two residues previously implicated in F-actin-binding form a buried salt-bridge between the N and C-terminal subdomains. The rest of the identified actin-binding residues are solvent-exposed and map onto a unique F-actin-binding surface.