Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

Structural genomics: beyond the human genome project.

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

Aromatic-aromatic interaction: a mechanism of protein structure stabilization.

Analysis of neighboring aromatic groups in four biphenyl peptides or peptide analogs and 34 proteins reveals a specific aromatic-aromatic interaction. Aromatic pairs (less than 7 A between phenyl ring centroids) were analyzed for the frequency of pair type, their interaction geometry (separation and dihedral angle), their nonbonded interaction energy, the secondary structural locations of interacting residues, their environment, and their conservation in related molecules. The results indicate that on average about 60 percent of aromatic side chains in proteins are involved in aromatic pairs, 80 percent of which form networks of three or more interacting aromatic side chains. Phenyl ring centroids are separated by a preferential distance of between 4.5 and 7 A, and dihedral angles approaching 90 degrees are most common. Nonbonded potential energy calculations indicate that a typical aromatic-aromatic interaction has energy of between -1 and -2 kilocalories per mole. The free energy contribution of the interaction depends on the environment of the aromatic pair. Buried or partially buried pairs constitute 80 percent of the surveyed sample and contribute a free energy of between -0.6 and -1.3 kilocalories per mole to the stability of the protein's structure at physiologic temperature. Of the proteins surveyed, 80 percent of these energetically favorable interactions stabilize tertiary structure, and 20 percent stabilize quaternary structure. Conservation of the interaction in related molecules is particularly striking.

Weight-reducing effects of the plasma protein encoded by the obese gene.

The gene product of the ob locus is important in the regulation of body weight. The ob product was shown to be present as a 16-kilodalton protein in mouse and human plasma but was undetectable in plasma from C57BL/6J ob/ob mice. Plasma levels of this protein were increased in diabetic (db) mice, a mutant thought to be resistant to the effects of ob. Daily intraperitoneal injections of either mouse or human recombinant OB protein reduced the body weight of ob/ob mice by 30 percent after 2 weeks of treatment with no apparent toxicity but had no effect on db/db mice. The protein reduced food intake and increased energy expenditure in ob/ob mice. Injections of wild-type mice twice daily with the mouse protein resulted in a sustained 12 percent weight loss, decreased food intake, and a reduction of body fat from 12.2 to 0.7 percent. These data suggest that the OB protein serves an endocrine function to regulate body fat stores.

Engaging the ribosome: universal IFs of translation.

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.

Structural aspects of interactions within the Myc/Max/Mad network.

Recently determined structures of a number of Myc family proteins have provided significant insights into the molecular nature of complex assembly and DNA binding. These structures illuminate the details of specific interactions that govern the assembly of nucleoprotein complexes and, in doing so, raise more questions regarding Myc biology. In this review, we focus on the lessons provided by these structures toward understanding (1) interactions that govern transcriptional repression by Mad via the Sin3 pathway, (2) homodimerization of Max, (3) heterodimerization of Myc-Max and Mad-Max, and (4) DNA recognition by each of the Max-Max, Myc-Max, and Mad-Max dimers.

Physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors IF1 and IF2.

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

The TATA box binding protein.

The TATA box binding protein is required by all three eukaryotic RNA polymerases to correctly initiate the transcription of ribosomal, messenger, small nuclear and transfer RNAs. Since the first gene encoding a TATA box binding protein was cloned from Saccharomyces cerevisiae, it has been the object of considerable biochemical and genetic study. Substantial progress has recently been made on structural and mechanistic studies of the protein. Three-dimensional structures newly elucidated include two TATA box binding proteins alone and bound to distinct TATA elements, and the ternary complex of transcription factor IIB recognizing a TATA box binding protein bound to a TATA element.

Winged helix proteins.

The winged helix proteins constitute a subfamily within the large ensemble of helix-turn-helix proteins. Since the discovery of the winged helix/fork head motif in 1993, a large number of topologically related proteins with diverse biological functions have been characterized by X-ray crystallography and solution NMR spectroscopy. Recently, a winged helix transcription factor (RFX1) was shown to bind DNA using unprecedented interactions between one of its eponymous wings and the major groove. This surprising observation suggests that the winged helix proteins can be subdivided into at least two classes with radically different modes of DNA recognition.

Histone-like transcription factors in eukaryotes.

Histone proteins have long been recognized as important regulators of eukaryotic gene expression. Condensation of DNA into chromatin by the core (H2A, H2B, H3, H4) and linker (H1, H5) histones effectively represses transcription initiation from the promoters of genes that have been packaged. Recently, eukaryotic transcriptional activators and coactivators (both positive and negative) resembling core and linker histone proteins have been discovered. Substantial progress has been made on structural and mechanistic studies of histones and histone-like transcription factors. Three-dimensional structures solved include the core histone octamer, an archael histone homodimer, two core histone-like subunits of transcription factor IID, a linker histone, and a linker histone-like transcriptional activator.

p53: a cellular Achilles' heel revealed.

Many human cancers result from the inactivation of p53, a protein central to DNA repair. Recent papers reporting the structures of two p53 domains help to rationalize the wealth of information about this protein.

PCD/DCoH: more than a second molecular saddle.

Two recent crystal structures of the tetrameric pterin-4 alpha-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF-1 (DCoH) protein provide fresh insights into how this multifunctional enzyme/transcriptional coactivator works.

Co-crystal structure of sterol regulatory element binding protein 1a at 2.3 A resolution.

BACKGROUND: The sterol regulatory element binding proteins (SREBPs) are helix-loop-helix transcriptional activators that control expression of genes encoding proteins essential for cholesterol biosynthesis/uptake and fatty acid biosynthesis. Unlike helix-loop-helix proteins that recognize symmetric E-boxes (5'-CANNTG-3'), the SREBPs have a tyrosine instead of a conserved arginine in their basic regions. This difference allows recognition of an asymmetric sterol regulatory element (StRE, 5'-ATCACCCAC-3'). RESULTS: The 2.3 A resolution co-crystal structure of the DNA-binding portion of SREBP-1a bound to an StRE reveals a quasi-symmetric homodimer with an asymmetric DNA-protein interface. One monomer binds the E-box half site of the StRE (5'-ATCAC-3') using sidechain-base contacts typical of other helix-loop-helix proteins. The non-E-box half site (5'-GTGGG-3') is recognized through entirely different protein-DNA contacts. CONCLUSIONS: Although the SREBPs are structurally similar to the E-box-binding helix-loop-helix proteins, the Arg-->Tyr substitution yields dramatically different DNA-binding properties that explain how they recognize StREs and regulate expression of genes important for membrane biosynthesis.

Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.

HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria.

The X-ray crystal structure of the Escherichia coli stress response protein HDEA has been determined at 2.0 A resolution. The single domain alpha-helical protein is found in the periplasmic space, where it supports an acid resistance phenotype essential for infectivity of enteric bacterial pathogens, such as Shigella and E. coli. Functional studies demonstrate that HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins. We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions.

Re-refinement of the X-ray crystal structure of bovine lens leucine aminopeptidase complexed with bestatin.

Bestatin, (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine, has been incorrectly modelled in the previously reported structure of the complex between bovine lens leucine aminopeptidase (blLAP) and bestatin. In the previously reported structure, the C2 of bestatin was modelled and refined in the R configuration instead of the correct S configuration. The structure of the blLAP-bestatin complex has been re-refined after remodelling bestatin in its correct stereochemistry.

Structure determination and refinement of bovine lens leucine aminopeptidase and its complex with bestatin.

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0 A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, this structure and the structure of the isomorphous native enzyme have been refined at 2.25 and 2.32 A resolution, respectively, with crystallographic R-factors of 0.180 and 0.159, respectively. The current structural model for the enzyme includes the two zinc ions and 481 of the 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry, and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. Monomers are crystallographically equivalent. Each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 A x 55 A x 55 A. The secondary structural composition is 35% alpha-helix and 23% beta-strand. The N-terminal domain (160 amino acid residues) mediates trimer-trimer interactions and does not appear to participate directly in catalysis, while the C-terminal domain (327 amino acid residues) is responsible for catalysis and binds the two zinc ions, which are less than 3 A apart. These two metal ions are located near the edge of an eight-stranded, saddle-shaped beta-sheet. The zinc ion that has the lower temperature factor is co-ordinated by one carboxylate oxygen atom from each of Asp255, Asp332 and Glu334, and the carbonyl oxygen of Asp332. The other zinc ion, presumed to be readily exchangeable, is co-ordinated by one carboxylate oxygen atom of each of Asp273 and Glu334 and the side-chain amino group of Lys250. The active site also contains two positively charged residues, Lys262 and Arg336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the 2-fold symmetry axes. Bestatin binds to one of the active site zinc ions, and its phenylalanine and leucine side-chains occupy hydrophobic pockets adjacent to the active site. Finally, the relationship between bovine lens leucine aminopeptidase and the homologous enzyme pepA from Escherichia coli is discussed.

Thermodynamic analysis of the interaction between YY1 and the AAV P5 promoter initiator element.

BACKGROUND: We previously determined the co-crystal structure of the zinc finger region of transcription factor YY1 (YY1Delta) bound to the initiator element (Inr) of the adenoassociated virus (AAV) P5 gene promoter [Houbaviy, H.B. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13577-13582]. Our structure explained both binding specificity and the ability of YY1 to support specific, unidirectional transcription initiation. RESULTS: To further understand Inr recognition by YY1, we analyzed the YY1Delta-Inr interaction by isothermal titration calorimetry (ITC) and used limited proteolysis, DNase I footprinting and missing nucleoside experiments to show that YY1Delta and full-length YY1 (YY1WT) have indistinguishable DNA binding properties. CONCLUSIONS: YY1 binding occurs at an equilibrium dissociation constant (K(d)) of about 1 microM, and exhibits a large negative heat capacity change (DeltaC(p)). We analyzed the thermodynamic behavior of YY1Delta in terms of buried solvent-accessible surface area resulting from interaction of two rigid bodies, which could not explain our measured value of DeltaC(p). We must, therefore, postulate conformational changes in YY1 and/or the Inr or question the validity of current DeltaC(p) analysis methods for protein-DNA interactions.

Deposit3D: a tool for automating structure depositions to the Protein Data Bank.

Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.

Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry.

A simple biochemical method that combines enzymatic proteolysis and matrix-assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA-binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max--a member of the basic/helix-loop-helix/zipper family of DNA-binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max-specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100-fold reduction in cleavage rates. Over a 2-day period, both complete and partial proteolysis of the Max-DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N-terminal and helix-loop-helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N-terminals. Less protection is seen at the C-terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix-loop-helix protein Max, the present method appears well suited for a range of other structural biological applications.