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The eukaryotic transcription factor Pax5 or B -cell s pecific a ctivator p rotein (BSAP) is central to B-cell development and has been implicated in a large number of cellular malignancies resulting from loss- or gain-of-function mutations. In this study, we characterized the DNA-binding Paired domain (PD) of Pax5 in its free and DNA-bound forms using NMR spectroscopy. In isolation, the PD folds as two independent helical bundle subdomains separated by a conformationally disordered linker. The two subdomains differ in stability, with the C-terminal subdomain (CTD) being ~ 10-fold more protected from amide hydrogen exchange (HX) than the N-terminal subdomain (NTD). Upon binding DNA, the linker and an induced N-terminal β-hairpin become ordered with significantly dampened motions and increased HX protection. Both subdomains of the PD contribute to specific DNA binding, resulting in an equilibrium dissociation constant more than three orders of magnitude lower than exhibited by the separate subdomains for their respective half-sites (nM versus μM). The isolated CTD binds non-specific DNA sequences with only ~ 10-fold weaker affinity than cognate sequences. In contrast, the NTD associates very poorly with non-specific DNA. We propose that the more stable CTD has evolved to provide relatively low affinity non-specific contacts with DNA. In contrast, the more dynamic NTD discriminates between cognate and non-specific sites. The distinct roles of the PD subdomains may enable efficient searching of genomic DNA by Pax5 while retaining specificity for functional regulatory sites. [ABSTRACT FROM AUTHOR]

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. [ABSTRACT FROM AUTHOR]

DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its Nand/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case, binding was mediated by hydrophobic and electrostatic interactions and weakened with increasing ionic strength. Neither isolated SIM showed any significant paralog specificity, and the measured μM range KD values of SIM-N toward both SUMO-1 and SUMO-2 were ∼4-fold lower than those of SIM-C. Furthermore, SIM-N bound SUMO-1 predominantly in a parallel orientation, whereas SIM-C interconverted between parallel and antiparallel binding modes on an ms to μs time scale. The differences in affinities and binding modes are attributed to the differences in charged residues that flank the otherwise identical hydrophobic core sequences of the two SIMs. In addition, within its native context, SIM-N bound intramolecularly to the adjacent N-terminal helical bundle domain of DAXX, thus reducing its apparent affinity for SUMO. This behavior suggests a possible autoregulatory mechanism for DAXX. The interaction of a C-terminal fragment of DAXX with an N-terminal fragment of the sumoylated Etsi transcription factor was mediated by SIM-C. Importantly, this interaction did not involve any direct contacts between DAXX and Etsi, but rather was derived from the non-covalent binding of SIM-C to SUMO-1, which in turn was covalently linked to the unstructured N-terminal segment of Etsi. These results provide insights into the binding mechanisms and hence biological roles of the DAXX SUMO-interacting motifs. [ABSTRACT FROM AUTHOR]

Summary: Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553–570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked β-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring. [Copyright &y& Elsevier]

Early NMR structural studies of serum lipoproteins were based on [sup 1]H,[sup 13]C,[sup 31]P, and[sup 2]H studies of lipid components. From the early studies information on composition, lipid chain dynamics and order parameters, and monolayer organization resulted. More recently, selective or complete isotopic labeling techniques, combined with multidimensional NMR spectroscopy, have resulted in structural information of apoprotein fragments. Finally, use of heteronuclear three- and four-dimensional experiments have yielded solution structures and protein-lipid interactions of intact apolipoproteins C-I, C-II, and A-I. [ABSTRACT FROM AUTHOR]

The type III secretion system (T3SS) is a multi-membrane-spanning protein channel used by Gram-negative pathogenic bacteria to secrete effectors directly into the host cell cytoplasm. In the many species reliant on the T3SS for pathogenicity, proper assembly of the outer membrane secretin pore depends on a diverse family of lipoproteins called pilotins. We present structural and biochemical data on the Salmonella enterica pilotin InvH and the S domain of its cognate secretin InvG. Characterization of InvH by X-ray crystallography revealed a dimerized, α-helical pilotin. Size-exclusion-coupled multi-angle light scattering and small-angle X-ray scattering provide supporting evidence for the formation of an InvH homodimer in solution. Structures of the InvH-InvG heterodimeric complex determined by X-ray crystallography and NMR spectroscopy indicate a predominantly hydrophobic interface. Knowledge of the interaction between InvH and InvG brings us closer to understanding the mechanisms by which pilotins assemble the secretin pore. • The pilotin InvH forms an α-helical homodimer when crystallized and in solution • The secretin InvG binds InvH in a 1:1 ratio along a hydrophobic groove • InvH homodimerizes and binds InvG using the same interface The type III secretion system is a virulence nanomachine used by pathogenic bacteria. Its outer-membrane secretin pore is assembled by a cognate pilotin lipoprotein. The Salmonella pilotin InvH uses a hydrophobic groove to homodimerize and to bind the secretin InvG. The interface provides a basis for future drug design. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis α-1,4-galactosyltransferase C (LgtC) is responsible for the transfer of α-galactose from donor UDP-galactose to the lipooligosaccharide terminal acceptor lactose. Crystal structures of its substrate analogue complexes have provided key insights into the galactosyl transfer mechanism, including a hypothesized need for active site mobility. Accordingly, we have used nuclear magnetic resonance spectroscopy to probe the structural dynamics of LgtC in its apo form and with bound substrate analogues. More than the expected number of signals were observed in the methyl-TROSY spectra of apo LgtC, indicating that the protein adopts multiple conformational states. Magnetization transfer experiments showed that the predominant states, termed "a" and "b", are in equilibrium on a time scale of seconds. Their relative populations change with temperature and mutations, and only the "b" state is competent for substrate binding. For both states, relaxation dispersion studies also revealed substantial millisecond time scale motions of isoleucine side chains within and distal to the active site. Although altered, these motions were still detected in LgtC with a noncovalently bound donor analogue. A mutant, LgtC-Q189E, which forms an unexpected glycosyl-enzyme intermediate via a residue (Asp190) distal from its active site, was also investigated. Apo LgtC-Q189E did not show any enhanced motions that might account for the dramatic structural change required for the galactosylation of Asp190, yet formation of a trapped glycosyl-enzyme intermediate substantially reduced its millisecond time scale conformational mobility. Although further studies are required to link the detected motions of LgtC with its enzymatic mechanism, this work clearly demonstrates the complex structural dynamics of a model glycosyltransferase. [ABSTRACT FROM AUTHOR]

Abstract: ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS domain. The C-terminal inhibitory domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and 15N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors. [Copyright &y& Elsevier]

Although sumoylation regulates a diverse and grow- ing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop con- taining the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as ‘beads-on-a-string,’ connected by a flexible isopeptide tether. Ac- cordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries. [ABSTRACT FROM AUTHOR]

Abstract: DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5′GGAA3′ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5′GGAA3′ versus 5′GGAT3′. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors. [Copyright &y& Elsevier]

The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a β-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants. generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within β-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Rex changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis. [ABSTRACT FROM AUTHOR]

Summary The Mycobacterium tuberculosis ATP-binding cassette transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). The structures of FHA-1 and FHA-2 were determined by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, respectively. Relative to the canonical 11-strand β-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one β-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (∼50 μM versus 500 μM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. We hypothesize that this enables tunable phosphorylation-dependent multimerization to regulate Rv1747 transporter activity. [ABSTRACT FROM AUTHOR]

The article investigates the lysine amines in CexCD and the trapped glycosyl-enzyme intermediate with the use of NMR spectroscopy to determine the ionization states of active site residues. It demonstrates that K47 interacts with the cellobiosyl moiety though its positively charged side chain and that CexCD has 18 lysines exposed on the surface of the protein. Amines that remain undetected in NMR was because of the optimization of spectral parameters for amides.