Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.     

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.     

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.     

15N NMR relaxation studies of backbone dynamics in free and steroid-bound Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)). The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.     

Internal dynamics of the tryptophan repressor (TrpR) and two functionally distinct TrpR variants, L75F-TrpR and A77V-TrpR, in their l-Trp-bound forms

Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo (l-tryptophan corepressor-bound) form have been characterized using (15)N nuclear magnetic resonance (NMR) relaxation. The three proteins possess very similar structures, ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific (15)N-T(1), (15)N-T(2), (15)N-{(1)H} nuclear Overhauser effect, reduced spectral density, and generalized order (S(2)) parameters indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins' DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e., helices A-C and F), and a comparable "stiffening" is observed for residues in the DNA recognition helix (helix E) of the helix D-turn-helix E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA binding, and molecular recognition of target operators. Comparison of the (15)N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicates that the single-point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways and are most pronounced in the apo forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.     

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.     

Structure and stability of an acidic fibroblast growth factor from Notophthalmus viridescens

The three-dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notophthalmus viridescens) is determined using multidimensional NMR techniques. Complete assignment of all the atoms ((1)H, (15)N, and (13)C) has been achieved using a variety of triple resonance experiments. 50 structures were calculated using hybrid distance geometry-dynamical simulated annealing technique with a total of 1359 constraints. The atomic root mean square distribution for the backbone atoms in the structured region is 0.60 A. The secondary structural elements include 12 beta-strands arranged antiparallely into a beta-barrel structure. The protein (nFGF-1) exists in a monomeric state upon binding to the ligand, sucrose octa sulfate (SOS), in a stoichiometric ratio of 1:1. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. The conformational stabilities of nFGF-1 and its structural and functional homologue from the human source (hFGF-1) are drastically different. The differential stabilities of nFGF-1 and hFGF-1 are attributed to the differences in the number of hydrogen bonds and the presence of solvent inaccessible cavities in the two proteins.     

Backbone dynamics of barstar: a (15)N NMR relaxation study

Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s(-1), indicating the presence of conformational averaging motions only for a small subset of residues.     

Application of cross-correlated NMR spin relaxation to the zinc-finger protein CRP2(LIM2): evidence for collective motions in LIM domains

The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.     

Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban

Phospholamban (PLB) is a 52 amino acid membrane-endogenous regulator of the sarco(endo)plasmic calcium adenosinetriphosphatase (SERCA) in cardiac muscle. PLB's phosphorylation and dephosphorylation at S16 modulate its regulatory effect on SERCA by an undetermined mechanism. In this paper, we use multidimensional (1)H/(15)N solution NMR methods to establish the structural and dynamics basis for PLB's control of SERCA upon S16 phosphorylation. For our studies, we use a monomeric, fully active mutant of PLB, where C36, C41, and C46 have been mutated to A36, F41, and A46, respectively. Our data show that phosphorylation disrupts the "L-shaped" structure of monomeric PLB, causing significant unwinding of both the cytoplasmic helix (domain Ia) and the short loop (residues 17-21) connecting this domain to the transmembrane helix (domains Ib and II). Concomitant with this conformational transition, we also find pronounced changes in both the pico- to nanosecond and the micro- to millisecond time scale dynamics. The (1)H/(15)N heteronuclear NOE values for residues 1-25 are significantly lower than those of unphosphorylated PLB, with slightly lower NOE values in the transmembrane domain, reflecting less restricted motion throughout the whole protein. These data are supported by the faster spin-lattice relaxation rates (R(1)) present in both the cytoplasmic and loop regions and by the enhanced spin-spin transverse relaxation rates (R(2)) observed in the transmembrane domain. These results demonstrate that while S16 phosphorylation induces a localized structural transition, changes in PLB's backbone dynamics are propagated throughout the protein backbone. We propose that the regulatory mechanism of PLB phosphorylation involves an order-to-disorder transition, resulting in a decrease in the PLB inhibition of SERCA.     

Conformational flexibility of a human immunoglobulin light chain variable domain by relaxation dispersion nuclear magnetic resonance spectroscopy: implications for protein misfolding and amyloid assembly

The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide (15)N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, (15)N Carr--Purcell--Meiboom--Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5-20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ~2-4% range. Upon acidification, the minor conformer populations increase significantly, to ~10-15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.     

Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C

Binding of Ca(2+) to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein-protein interactions that initiate contraction. Cardiac TnC contains two Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C(p,j)) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol(-1) K(-1). The average heat capacity for Ca(2+) binding site 1 is larger than that for site 2 by 1.3 +/- 0.8 cal mol(-1) K(-1), and likely represents a mechanism where differences in affinity between Ca(2+) binding sites for EF hand proteins can be modulated.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

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.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.     

Correlation of binding-loop internal dynamics with stability and function in potato I inhibitor family: relative contributions of Arg(50) and Arg(52) in Cucurbita maxima trypsin inhibitor-V as studied by site-directed mutagenesis and NMR spectroscopy

The side chains of Arg(50) and Arg(52) at positions P(6)' and P(8)', respectively, anchor the binding loop to the protein scaffold by means of hydrogen bonds in Cucurbita maxima trypsin inhibitor-V (CMTI-V), a potato I family member. Here, we have investigated the relative contributions of Arg(50) and Arg(52) to the binding-loop flexibility and stability by determining changes in structure, dynamics, and proteolytic stability as a consequence of individually mutating them into an alanine. We have compared chemical shift assignments of main-chain hydrogens and nitrogens, and (1)H-(1)H interresidue nuclear Overhauser effects (NOEs) for the two mutants with those of the wild-type protein. We have also measured NMR longitudinal and transverse relaxation rates and (15)N-(1)H NOE enhancements for all backbone and side-chain NH groups and calculated the model-free parameters for R50A-rCMTI-V and R52A-rCMTI-V. The three-dimensional structures and backbone dynamics of the protein scaffold region remain very similar for both mutants, relative to the wild-type protein. The flexibility of the binding loop is increased in both R50A- and R52A-rCMTI-V. In R52A-rCMTI-V, the mean generalized order parameter () of the P(6)-P(1) residues of the binding loop (39-44) decreases to 0.68 +/- 0.02 from 0.76 +/- 0.04 observed for the wild-type protein. However, in R50A-rCMTI-V, the flexibility of the whole binding loop increases, especially that of the P(1)'-P(3)' residues (45-47), whose value drops dramatically to 0.35 +/- 0.03 from 0.68 +/- 0.03 determined for rCMTI-V. More strikingly, S(2) values of side-chain N epsilon Hs reveal that, in the R50A mutant, removal of the R50 hydrogen bond results in the loss of the R52 hydrogen bond too, whereas in R52A, the R50 hydrogen bond remains unaffected. Kinetic data on trypsin-catalyzed hydrolysis of the reactive-site peptide bond (P(1)-P(1)') suggest that the activation free energy barrier of the reaction at 25 degrees C is reduced by 2.1 kcal/mol for R50A-rCMTI-V and by 1.5 kcal/mol for R52A-rCMTI-V, relative to rCMTI-V. Collectively, the results suggest that although both the P(6') and P(8)' anchors are required for optimal inhibitor function and stability in the potato I family, the former is essential for the existence of the latter and has greater influence on the binding-loop structure, dynamics, and stability.     

Guanine nucleotides differentially modulate backbone dynamics of the STAS domain of the SulP/SLC26 transport protein Rv1739c of Mycobacterium tuberculosis

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (μs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.     

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.