Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.     

Secondary Structure and Molecular Evolution of the Mitochondrial Small Subunit Ribosomal RNA in Agaricales (Euagarics clade,Homobasidiomycota)

The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA core is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3 end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.     

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.     

Dissecting Drosophila embryonic brain development using photoactivated gene expression

The Drosophila brain is generated by a complex series of morphogenetic movements. To better understand brain development and to provide a guide for experimental manipulation of brain progenitors, we created a fate map using photoactivated gene expression to mark cells originating within specific mitotic domains and time-lapse microscopy to dynamically monitor their progeny. We show that mitotic domains 1, 5, and 9 give rise to discrete cell populations within specific regions of the brain. Two novel observations were that the antennal sensory system, composed of four disparate cell clusters, arose from mitotic domain 5 and that mitotic domain B produced glial cells, while neurons were produced from mitotic domains 1, 5, and 9. Time-lapse analysis of marked cells showed complex mitotic and migratory patterns for cells derived from these mitotic domains. Photoactivated gene expression was also used either to kill, to induce ectopic divisions, or to alter cell fate. This revealed that deficits were not repopulated, while ectopic cells were removed and extra glia were tolerated.     

The DNA topoisomerase I binding protein topors as a novel cellular target for SUMO-1 modification: characterization of domains necessary for subcellular localization and sumolation

Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles.     

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.     

Isochore chromosome maps of the human genome

The human genome is a mosaic of isochores, which are long DNA segments (300 kbp) relatively homogeneous in G+C. Human isochores were first identified by density-gradient ultracentrifugation of bulk DNA, and differ in important features, e.g. genes are found predominantly in the GC-richest isochores. Here, we use a reliable segmentation method to partition the longest contigs in the human genome draft sequence into long homogeneous genome regions (LHGRs), thereby revealing the isochore structure of the human genome. The advantages of the isochore maps presented here are: (1) sequence heterogeneities at different scales are shown in the same plot; (2) pair-wise compositional differences between adjacent regions are all statistically significant; (3) isochore boundaries are accurately defined to single base pair resolution; and (4) both gradual and abrupt isochore boundaries are simultaneously revealed. Taking advantage of the wide sample of genome sequence analyzed, we investigate the correspondence between LHGRs and true human isochores revealed through DNA centrifugation. LHGRs show many of the typical isochore features, mainly size distribution, G+C range, and proportions of the isochore classes. The relative density of genes, Alu and long interspersed nuclear element repeats and the different types of single nucleotide polymorphisms on LHGRs also coincide with expectations in true isochores. Potential applications of isochore maps range from the improvement of gene-finding algorithms to the prediction of linkage disequilibrium levels in association studies between marker genes and complex traits. The coordinates for the LHGRs identified in all the contigs longer than 2 Mb in the human genome sequence are available at the online resource on isochore mapping: http://bioinfo2.ugr.es/isochores.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.