Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Crystallization and preliminary X-ray diffraction analysis of GCIP/HHM transcriptional regulator.

GCIP/HHM is a human nuclear protein that is implicated in regulation of cell proliferation. Its primary structure contains helix-loop-helix and leucine-zipper motifs but lacks a DNA-binding basic region. Native and selenomethionine-derivatized (SeMet) crystals of full-length GCIP/HHM were obtained using the hanging-drop vapour-diffusion method. The crystals were greatly improved by adding tris(2-carboxyethyl)phosphine as a reducing reagent and diffracted to 3.5 A resolution. Preliminary phase calculations using the data set obtained from the SeMet crystal suggested that the crystal belonged to space group P3(2)21 and contained one molecule per asymmetric unit. Structure determination by the multiple-wavelength anomalous dispersion method using the SeMet crystals is in progress.

Molecular basis for SUMOylation-dependent regulation of DNA binding activity of heat shock factor 2.

Heat shock factor 2 (HSF2) is a member of a vertebrate transcription factor family for genes of heat shock proteins and is involved in the regulation of development and cellular differentiation. The DNA binding property of HSF2 is modulated by the post-translational modification of a specific lysine residue in its DNA binding domain by small ubiquitin-like modifier (SUMO), but the consequences of SUMOylation and its underlying molecular mechanism remain unclear. Here we show the inhibitory effect of SUMOylation on the interaction between HSF2 and DNA based on biochemical analysis using isolated recombinant HSF2. NMR study of the SUMOylated DNA binding domain of HSF2 indicates that the SUMO moiety is flexible with respect to the DNA binding domain and has neither a noncovalent interface with nor a structural effect on the domain. Combined with data from double electron-electron resonance and paramagnetic NMR relaxation enhancement experiments, these results suggest that SUMO attachment negatively modulates the formation of the protein-DNA complex through a randomly distributed steric interference.

The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation.

The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.

ATP-induced structural change of dephosphocoenzyme A kinase from Thermus thermophilus HB8.

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.

Crystallographic characterization of the N-terminal domain of PEX1.

Peroxisomal enzymes are responsible for several primary metabolism pathways, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases and both are necessary for the import of more than 50 peroxisomal resident proteins from the cytosol into peroxisomes. In this study, PEX1 N-terminal domain crystals have been prepared. The crystals belong to space group P3(1) or P3(2), with unit-cell parameters a = b = 63.5 A, c = 33.5 A, and contain one protein molecule per crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.05 A.

Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain.

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.

Structural basis for neurofibromatosis type 2. Crystal structure of the merlin FERM domain.

Neurofibromatosis type 2 (NF2) is a dominantly inherited disease associated with the central nervous system. The NF2 gene product merlin is a tumor suppressor, and its mutation or inactivation causes this disease. We report here the crystal structure of the merlin FERM domain containing a 22-residue alpha-helical segment. The structure reveals that the merlin FERM domain consists of three subdomains displaying notable features of the electrostatic surface potentials, although the overall surface potentials similar to those of ezrin/radixin/moesin (ERM) proteins indicate electrostatic membrane association. The structure also is consistent with inactivation mechanisms caused by the pathogenic mutations associated with NF2.