Dynamics of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

A significant determinant for the broad substrate specificity of the metallo-beta-lactamases from Bacteroides fragilis and other similar organisms is the presence of a plastic substrate binding site that is nevertheless capable of tight substrate binding in the Michaelis complex. To achieve these two competing ends, the molecule apparently employs a flexible flap that closes over the active site in the presence of substrate. These characteristics imply that dynamic changes are an important component of the mechanism of action of these enzymes. The backbone and tryptophan side chain dynamics of the metallo-beta-lactamase from B. fragilis have been examined using (15)N NMR relaxation measurements. Two states of the protein were examined, in the presence and absence of a tight-binding inhibitor. Relaxation measurements were analyzed by the model-free method. Overall, the metallo-beta-lactamase molecule is rigid and shows little flexibility except in loops. The flexibility of the loop that covers the active site is not unusually great as compared to the other loops of the protein. Local motion on a picosecond time scale was found to be very similar throughout the protein in the presence and absence of the inhibitor, but a significant difference was observed in the motions on a nanosecond time scale (tau(e)). Large-amplitude motions with a time constant of about 1.3 ns were observed for the flexible flap region (residues 45-55) in the absence of the inhibitor. These motions were completely damped out in the presence of the inhibitor. In addition, the motion of a tryptophan side chain at the tip of the beta-hairpin of the flap shows a very significant difference in motion on the ps time scale. These results indicate that the motions of the polypeptide chain in the flap region can be invoked to explain both the wide substrate specificity (the free form has considerable amplitude of motion in this region) and the catalytic efficiency of the metallo-beta-lactamase (the motions are damped out when the inhibitor and by implication a substrate binds in the active site).     

Role of a solvent-exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo-beta-lactamase

Numerous X-ray crystal structures of the metallo-beta-lactamase from Bacteroides fragilis and related organisms show a beta-hairpin loop immediately adjacent to the active-site zinc atom(s). Both crystallographic and NMR information show that the end of this beta-hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X-ray structures to a lack of observable electron density in this region. We report an investigation of the role of this mobile, solvent-exposed tryptophan using site-directed mutagenesis, steady state kinetics measurements and characterization by NMR. Trp49 appears to have a role both in substrate binding and in promotion of catalysis. Substitution of this residue with a number of different side chains indicates that the binding interaction depends on the bulky hydrophobic and aromatic nature of the indole ring, which can provide relatively non-specific interactions with a variety of antibiotic substrates. In this way, the tryptophan at this position provides a large degree of the breadth of substrate specificity for the metallo-beta-lactamase. Previous studies established that the antibiotic binding site was sufficiently plastic that the derivatization of existing antibiotics is unlikely to result in the successful treatment of bacterial infections incorporating this resistance element. Rather, a more productive approach may be to design therapeutics directed towards this solvent-exposed tryptophan residue.     

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.     

Backbone (15)N relaxation analysis of the N-terminal domain of the HTLV-I capsid protein and comparison with the capsid protein of HIV-1

Human T-cell leukemia virus type 1 (HTLV-I) is an oncogenic retrovirus that exhibits specific tropism for human T-cells. The capsid (CA) proteins of retroviruses share highly conserved secondary and tertiary structures. However, they can form quaternary structures (assembled cores) that are conical (e.g., the lentivirus subgroup, including HIV) or spherical (e.g., the oncovirus subgroup, including HTLV). The intrinsic features that drive these differences are not understood. So far, only structural studies have been used as a basis for comparison. Dynamics may play a role in particle formation. High-resolution nuclear magnetic resonance (NMR) (15)N relaxation data (T(1), T(1rho), and NOE) have been used to characterize the backbone dynamics of the N-terminal domain (NTD) of the oncovirus HTLV-I and to compare with the CA NTD of HIV-1. Large variations in the (15)N heteronuclear NOEs and transversal relaxation rates for individual residues are consistent with the bundle RMSD of the previously calculated NMR structures. The beta-hairpin and CyP-A loop exhibit different mobility in HTLV-I and HIV-1. The overall hydrodynamic property of the HTLV-I capsid NTD is quite distinct from the HIV-1.     

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.     

Backbone 1H, 13C, and 15N assignments for the tandem ubiquitin binding domains of signal transducing adapter molecule 1

Signal transducing adapter molecule (STAM) forms the endosomal sorting complex required for transport-0 (ESCRT-0) complex with hepatocyte growth factor-regulated substrate (Hrs) to sort the ubiquitinated cargo proteins from the early endosomes to the ESCRT-1 complex. ESCRT-0 complex, STAM and Hrs, contains multiple ubiquitin binding domains, in which STAM has two ubiquitin binding domains, Vps27/Hrs/Stam (VHS) and ubiquitin interacting motif (UIM) at its N-terminus. By the cooperation of the multiple ubiquitin binding domains, the ESCRT-0 complex recognizes poly-ubiquitin, especially Lys63-linked ubiquitin. Here, we report the backbone resonance assignments and the secondary structure of the N-terminal 191 amino acids of the human STAM1 which includes the VHS domain and UIM. The {¹H}-¹⁵N heteronuclear NOE experiments revealed that an unstructured and flexible loop region connects the VHS domain and UIM. Our work provides the basic information for the further NMR investigation of the interaction between STAM1 and poly-ubiquitin.     

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from<Emphasis Type="Italic"> Ralstonia metallidurans</Emphasis> CH34

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. a has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTCXXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl–methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. ¹⁵N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first -strand and the first -helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0495-yAbbreviations CCA -cyano-4-hydroxy-trans-cinnamic acid - CSI chemical shift index - HSQC ¹H-detected heteronuclear single-quantum coherence - MerAa the 68 amino acid N-terminal extension of the mercuric reductase - NOE nuclear Overhauser effect - RDCs residual dipolar couplings - TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride     

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.     

NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.     

A comparative study of the backbone dynamics of two closely related lipid binding proteins: Bovine heart fatty acid binding protein and porcine ileal lipid binding protein

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the model-free approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the sec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.     

Structural and dynamics studies of the D54A mutant of human T cell leukemia virus-1 capsid protein

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.     

Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR

Summary The Rev Response Element (RRE) RNA-Rev protein interaction is important for regulation of gene expression in the human immunodeficiency virus. A model system for this interaction, which includes stem IIB of the RRE RNA and an arginine-rich peptide from the RNA-binding domain of Rev, was studied using multidimensional heteronuclear NMR. Assignment of the RNA when bound to the peptide was obtained from NMR experiments utilizing uniformly and specifically ¹³C-labeled RNA. Isotopic filtering experiments on the specifically labeled RNA enabled unambiguous assignment of unusual nonsequential NOE patterns present in the internal loop of the RRE. A three-dimensional model of the RNA in the complex was obtained using restrained molecular dynamics calculations. The internal loop contains two purine-purine base pairs, which are stacked to form one continuous helix flanked by two A-form regions. The formation of a G-G base pair in the internal loop requires an unusual structure of the phosphate backbone. This structural feature is consistent with mutational data as being important for the binding of Rev to the RRE. The G-G base pair may play an important role in opening the normally narrow major groove of A-form RNA to permit binding of the Rev basic domain.     

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.     

Homologous proteins with different folds: the three-dimensional structures of domains 1 and 6 of the multiple Kazal-type inhibitor LEKTI

We have determined the solution structures of recombinant domain 1 and native domain 6 of the multi-domain Kazal-type serine proteinase inhibitor LEKTI using multi-dimensional NMR spectroscopy. While two of the 15 potential inhibitory LEKTI domains contain three disulfide bonds typical of Kazal-type inhibitors, the remaining 13 domains have only two of these disulfide bridges. Therefore, they may represent a novel type of serine proteinase inhibitor. The first and the sixth LEKTI domain, which have been isolated from human blood ultrafiltrate, belong to this group. In spite of sharing the same disulfide pattern and a sequence identity of about 35% from the first to the fourth cysteine, the two proteins show different structures in this region. The three-dimensional structure of domain 6 consists of two helices and a beta-hairpin structure, and closely resembles the three-dimensional fold of classical Kazal-type serine proteinase inhibitors including the inhibitory binding loop. Domain 6 has been shown to be an efficient, but non-permanent serine proteinase inhibitor. The backbone geometry of its canonical loop is not as well defined as the remaining structural elements, providing a possible explanation for its non-permanent inhibitory activity. We conclude that domain 6 belongs to a subfamily of classical Kazal-type inhibitors, as the third disulfide bond and a third beta-strand are missing. The three-dimensional structure of domain 1 shows three helices and a beta-hairpin, but the central part of the structure differs remarkably from that of domain 6. The sequence adopting hairpin structure in domain 6 exhibits helical conformation in domain 1, and none of the residues within the putative P3 to P3' stretch features backbone angles that resemble those of the canonical loop of known proteinase inhibitors. No proteinase has been found to be inhibited by domain 1. We conclude that domain 1 adopts a new protein fold and is no canonical serine proteinase inhibitor.     

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.     

Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface

Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/1H HSQC cross peaks were observed and 180 of these were assignable, indicating that approximately 30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least approximately 40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H-15N NOE experiments detected picosecond timescale backbone motion in the alphaF-beta9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was quenched after binding 13-PD. Collectively, these observations reveal that, in solution, Fpg is a very dynamic molecule even after binding damaged DNA. Such motion, especially at the DNA binding surface, may be key to its processive search for DNA damage and its catalytic functions once it recognizes damaged DNA.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.     

The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural beta-hairpin motif

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable beta-hairpin, incorporating a type VIb beta-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 A over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein beta-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide's solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation approximately 0.7 A) but also for the side-chains. This isolated beta-hairpin retains the biologically active "canonical conformation" typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this beta-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this beta-hairpin and its biological activity is discussed.     

Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins.

High-alkaline serine proteases have been successfully applied as protein degrading components of detergent formulations and are subject to extensive protein engineering efforts to improve their stability and performance. Dynamics has been suggested to play an important role in determining enzyme activity and specificity and it is therefore of interest to establish how local changes in internal mobility affect protein stability, specificity and performance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, that has improved washing performance. T1, T2 and heteronuclear NOE measurements of the 15N nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 and S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the catalytic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonstrate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop regions involved in substrate binding become more structured in the engineered variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been established by X-ray crystallograpic methods, as shown in the accompanying paper.     

Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding G(B1). We have assigned almost all backbone (15)N, H(N), and H(alpha) resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state ?(1)H-(15)N? NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.