Backbone dynamics of the c-Myb DNA-binding domain complexed with a specific DNA

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2R3 fragment. Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.     

The solution structure of the homeodomain of the rat insulin-gene enhancer protein isl-1. Comparison with other homeodomains.

Homeodomains are one of the key families of eukaryotic DNA-binding motifs and provide an important model system for DNA recognition. We have determined a high-quality nuclear magnetic resonance (NMR) structure of the DNA-binding homeodomain of the insulin gene enhancer protein Isl-1 (Isl-1-HD). It forms the first solution structure of a homeodomain from the LIM family. It contains a well-defined inner core (residues 12-55) consisting of the classical three-helix structure observed in other homeodomains. The N terminus is unstructured up to residue 8, while the C terminus gradually becomes unstructured from residue 55 onwards. Some flexibility is evident in the loop parts of the inner core. Isl-1-HD has, despite its low sequence identity (23-34 %), a structure that is strikingly similar to that of the other homeodomains with known three-dimensional structures. Detailed analysis of Isl-1-HD and the other homeodomains rationalizes the differences in their temperature stability and explains the low stability of the Isl-1-HD in the free state (tm 22-30 degrees C). Upon DNA binding, a significant stabilization occurs (tm>55 degrees C). The low stability of Isl-1-HD (and other mammalian homeodomains) suggests that in vivo Isl-1-HD recognizes its cognate DNA from its unfolded state.     

Insights into Lou Gehrig's disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase

We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.     

Assignments of backbone 1H, 13C and 15N resonances in H-Ras (1–166) complexed with GppNHp at physiological pH

The small GTPase Ras is an important signaling molecule acting as a molecular switch in eukaryotic cells. Recent findings of global conformational exchange and a putative allosteric binding site in the G domain of Ras opened an avenue to understanding novel aspects of Ras function. To facilitate detailed NMR studies of Ras in physiological solution conditions, we performed backbone resonance assignments of Ras bound to slowly hydrolysable GTP mimic, guanosine 5′-[ß, γ-imido]triphosphate at pH 7.2. Out of 163 non-proline residues of the G domain, signals from backbone amide proton, nitrogen and carbon spins of 127 residues were confidently assigned with the remaining unassigned residues mostly located at the exchange-broadened effectors interface.     

Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity

Mammalian DNA polymerase beta functions in the base excision DNA repair pathway filling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of beta-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5'-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RMSD of the NMR conformers (residues 13-80) is 0.37 A for the backbone heavy atoms and 0.78 A for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5'-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Omega-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (R(ex)) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Omega-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.     

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.     

Conformational dynamics and molecular recognition: backbone dynamics of the estrogen receptor DNA-binding domain.

We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.     

The three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex by NMR spectroscopy.

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly15N and doubly13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1' resonances. The DNA duplex is nearly straight and its structure is primarily that of B -DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three- dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.     

Backbone dynamics of the C-terminal SH2 domain of the p85alpha subunit of phosphoinositide 3-kinase: effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes

The backbone dynamics of the C-terminal SH2 domain from the regulatory subunit p85alpha (p85alpha C-SH2) of phosphoinositide 3-kinase has been investigated in the absence of, and in complex with, a high-affinity phosphotyrosine-containing peptide ligand derived from the platelet-derived growth-factor receptor. (15)N R(1) and R(2) relaxation rates and steady-state [(1)H]-(15)N NOE values were measured by means of (1)H-(15)N correlated two-dimensional methods and were analyzed within the framework of the model-free formalism. Several residues in the BC loop and in the neighbouring secondary structural elements display fast local dynamics in the absence of phosphotyrosine peptide ligand as evidenced by below-average [(1)H]-(15)N NOE values. Furthermore, residue Gln41 (BC3) displays conformational exchange phenomena as indicated by an above-average R(2) relaxation rate. Upon binding of the phosphotyrosine peptide, the NOE values increase to values observed for regular secondary structure and the exchange contribution to the R(2) relaxation rate for Gln41 (BC3) vanishes. These observations indicate a loss of backbone flexibility upon ligand binding. Substantial exchange contributions for His56 (betaD4) and Cys57 (betaD5), which are known to make important interactions with the ligand, are attenuated upon ligand binding. Several residues in the betaD'-FB region and the BG loop, which contribute to the ligand binding surface of the protein, exhibit exchange terms which are reduced or vanish when the ligand is bound. Together, these observations suggest that ligand binding is accompanied by a loss of conformational flexibility on the ligand binding face of the protein. However, comparison with other SH2 domains reveals an apparent lack of consensus in the changes in dynamics induced by ligand binding. Exchange rates for individual residues were quantified in peptide-complexed p85alpha C-SH2 from the dependence of the exchange contributions on the CPMG delay in an R(2) series and show that peptide-complexed p85alpha C-SH2 is affected by multiple conformational exchange processes with exchange rate constants from 10(2) s(-1) to 7.10(3) s(-1). Mapping of the exchange-rate constants on the protein surface show a clustering of residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process.     

Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the micros-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3(10)- and alpha-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.     

Murine interleukin-3: structure, dynamics, and conformational heterogeneity in solution

Interleukin-3 (IL-3), a cytokine produced primarily by activated T-cells during immune responses, is a crucial regulator of allergic inflammation. The three-dimensional structure of murine IL-3 (mIL-3) has remained elusive owing to its poor solubility and strong tendency toward aggregation under solution conditions typically used for structural studies. Here we describe the solution properties and structure of mIL-3 determined by NMR using an engineered construct of mIL-3 (mIL-3(33-156)). mIL-3 adopts a four-helical bundle fold, typical of proteins belonging to the short-chain cytokine family, and features a core of highly conserved hydrophobic residues. While significant line broadening and peak disappearance were observed in NMR spectra at higher temperatures, there was no evidence for temperature-dependent changes of the oligomeric state of mIL-3(33-156). Further analysis of the temperature dependence of amide (1)H chemical shifts and backbone (15)N relaxation parameters, including (15)N relaxation dispersion, revealed the presence of significant conformational exchange and local conformational heterogeneity. Residues recently shown by mutagenesis to play key roles in β(IL-3) receptor recognition and activation, which are located within the α(A) and α(C) helices and aligned on one face of the mIL-3(33-156) structure, are relatively rigid. In contrast, pronounced conformational heterogeneity was observed for a cluster of residues located on the opposite side of mIL-3, which corresponds spatially to sites in the related cytokines human IL-3, IL-5, and GM-CSF that are known to mediate interactions with their respective α-receptor subunits. Such conformational heterogeneity may facilitate the interaction of mIL-3 with each of two naturally occurring mIL-3Rα isoforms, leading to structurally distinct high-affinity complexes.     

Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis

The site-specific DNA recombinase, gammadelta resolvase, from Escherichia coli catalyzes recombination of res site-containing plasmid DNA to two catenated circular DNA products. The catalytic domain (residues 1-105), lacking a C-terminal dimerization interface, has been constructed and the NMR solution structure of the monomer determined. The RMSD of the NMR conformers for residues 2-92 excluding residues 37-45 and 64-73 is 0.41 A for backbone atoms and 0.88 A for all heavy atoms. The NMR solution structure of the monomeric catalytic domain (residues 1-105) was found to be formed by a four-stranded parallel beta-sheet surrounded by three helices. The catalytic domain (residues 1-105), deficient in the C-terminal dimerization domain, was monomeric at high salt concentration, but displayed unexpected dimerization at lower ionic strength. The unique solution dimerization interface at low ionic strength was mapped by NMR. With respect to previous crystal structures of the dimeric catalytic domain (residues 1-140), differences in the average conformation of active-site residues were found at loop 1 containing the catalytic S10 nucleophile, the beta1 strand containing R8, and at loop 3 containing D67, R68 and R71, which are required for catalysis. The active-site loops display high-frequency and conformational backbone dynamics and are less well defined than the secondary structures. In the solution structure, the D67 side-chain is proximal to the S10 side-chain making the D67 carboxylate group a candidate for activation of S10 through general base catalysis. Four conserved Arg residues can function in the activation of the phosphodiester for nucleophilic attack by the S10 hydroxyl group. A mechanism for covalent catalysis by this class of recombinases is proposed that may be related to dimer interface dissociation.