Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.

The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.     

Co-repressor induced order and biotin repressor dimerization: a case for divergent followed by convergent evolution

BirA catalyzes the adenylation and subsequent covalent attachment of biotin to the biotin carboxyl carrier protein (BCCP). In the absence of apo-BCCP, biotin-5'-AMP acts as a co-repressor that induces BirA dimerization and binding to the bio operator to repress biotin biosynthesis. The crystal structures of apo-BirA, and BirA in complex with biotin have been reported. We here describe the 2.8A resolution crystal structure of BirA in complex with the co-repressor analog biotinol-5'-AMP. It was previously shown that the structure of apo-BirA is monomeric and that binding of biotin weakly induces a dimeric structure in which three disordered surface loops become organized to form the dimer interface. The structure of the co-repressor complex is also a dimer, clearly related to the BirA.biotin structure, but with several significant conformational changes. A hitherto disordered "adenylate binding loop" forms a well-defined structure covering the co-repressor. The co-repressor buttresses the dimer interface, resulting in improved packing and a 12 degrees change in the hinge-bending angle along the dimer interface relative to the BirA.biotin structure. This helps explain why the binding of the co-repressor is necessary to optimize the binding of BirA to the bioO operator. The structure reveals an unexpected use of the nucleotide-binding motif GXGXXG in binding adenylate and controlling the repressor function. Finally, based on structural analysis we propose that the class of adenylating enzymes represented by BirA, lipoate protein ligase and class II tRNA synthetases diverged early and were selected based on their ability to sequester co-factors or amino acid residues, and adenylation activity arose independently through functional convergence.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

Multiple disordered loops function in corepressor-induced dimerization of the biotin repressor

Cooperative association of the Escherichia coli biotin repressor with the biotin operator is allosterically activated by binding of the corepressor, bio-5'-AMP. The corepressor function of the adenylate is due, in part, to its ability to induce repressor dimerization. Since a high-resolution structure of only the apo or unliganded repressor is currently available, the location of the dimerization interface on the protein structure is not known. Here, five mutants in the corepressor-binding domain of the repressor have been analyzed with respect to their DNA-binding and self-assembly properties. Results of these studies reveal that four of the mutant proteins exhibit defects in DNA binding. These same proteins are compromised in self-assembly. Furthermore, in the three-dimensional structure of the apo protein the mutations all lie in partially disordered surface loops, one of which is known to participate directly in corepressor binding. These results suggest that multiple disordered surface loops function in the corepressor-induced dimerization required for sequence-specific DNA binding by the biotin repressor.     

Sequence–function relationships in folding upon binding

Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5′-AMP (bio-5′-AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5′-AMP binding enhancing the dimerization free energy by −4.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.     

Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding

The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.     

Kinetics of loop formation and breakage in the denatured state of iso-1-cytochrome c

The earliest events in protein folding involve the formation of simple loops. Observing the rates of loop closure under denaturing conditions can provide direct insight into the relative probability and sequence determinants for formation of loops of different sizes. The persistence of these initial contacts is equally important for efficient folding, so measurement of rates of loop breakage under denaturing conditions is also essential. We have used stopped-flow and continuous-flow methods to measure the rates of histidine-heme loop formation and breakage in the denatured state of iso-1-cytochrome c (in the presence of 3 M guanidine HCl). The data indicate that the mechanism for forming loops is a two-step process, the first step being the deprotonation of the histidine, and the second step being the binding of the histidine to the heme. This mechanism makes it possible to extract both the rate constants of formation, k(f), and breakage, k(b), of loops from the pH dependence of the observed rate constant, k(obs). To determine the dependence of k(f) and k(b) on loop size, we have carried out kinetic measurements for seven single surface histidine variants of iso-1-cytochrome c. A scaling factor (the dependence of k(f) on log[loop size]) of approximately -1.8 is observed for loop formation, similar to that observed in other systems. The magnitude of k(b) varies from 30 s(-1) to 300 s(-1), indicating that the stability of different loops varies considerably. The implications of the kinetics of loop formation and breakage in the denatured state for the mechanism of protein folding are discussed.     

Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5′-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 Å resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg²⁺, and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation.     

mRNA环结构对蛋白质折叠速率的影响

前期的相关研究发现mRNA二级结构中存在对蛋白质折叠速率的重要影响因素.而mRNA二级结构中普遍存在着各种复杂的环结构,这些环结构是否对蛋白质折叠速率也有重要的影响呢?不同的环结构对蛋白质折叠速率的影响是否相同呢?基于此想法,建立了一个包含mRNA内部环、发夹环、膨胀环和多分支环等环结构信息和相应蛋白质折叠速率的数据库.对于数据库中的每一个蛋白质,计算了mRNA二级结构中各种环结构碱基含量、配对碱基含量及单链碱基含量等参量,分析了各参量与相应蛋白质折叠速率的相关性.结果显示,各种环结构碱基含量与蛋白质折叠速率均呈极显著或显著正相关.说明mRNA环结构对蛋白质折叠速率有重要的影响.进一步,把蛋白质按照不同折叠类型或不同二级结构类型分组后,对每一组蛋白质重复上述的分析工作.结果表明,对不同类蛋白质,mRNA的各种环结构对其相应蛋白质折叠速率的影响存在着显著差异.上述研究将为进一步开展有关mRNA和蛋白质折叠速率的研究奠定理论基础.