Influence of heme post-translational modification and distal ligation on the backbone dynamics of a monomeric hemoglobin

The cyanobacterium Synechococcus sp. PCC 7002 uses a hemoglobin of the truncated lineage (GlbN) in the detoxification of reactive species generated in the assimilation of nitrate. In view of a sensing or enzymatic role, several states of GlbN are of interest with respect to its structure-activity relationship. Nuclear magnetic resonance spectroscopy was applied to compare the structure and backbone dynamics of six GlbN forms differing in their oxidation state [Fe(II) or Fe(III)], distal ligand to the iron (histidine, carbon monoxide, or cyanide), or heme post-translational modification (b heme or covalently attached heme). Structural properties were assessed with pseudocontact shift calculations. (15)N relaxation data were analyzed by reduced spectral density mapping (picosecond to nanosecond motions) and by inspection of elevated R(2) values (microsecond to millisecond motions). On the picosecond to nanosecond time scale, GlbN exhibited little flexibility and was unresponsive to the differences among the various forms. Regions of slightly higher mobility were the CE turn, the EF loop, and the H-H' kink. In contrast, fluctuations on the microsecond to millisecond time scale depended on the form. Cyanide binding to the ferric state did not enhance motions, whereas reduction to the ferrous bis-histidine state resulted in elevated R(2) values for several amides. This response was attributed, at least in part, to a weakening of the distal histidine coordination. Carbon monoxide binding quenched some of these fluctuations. The results emphasized the role of the distal ligand in dictating backbone flexibility and illustrated the multiple ways in which motions are controlled by the hemoglobin fold.     

Backbone dynamics of a winged helix protein and its DNA complex at different temperatures: changes of internal motions in genesis upon binding to DNA.

The dynamic properties of a winged helix protein, Genesis, and its DNA complex at different temperatures were studied. Due to the complexity of motions, the commonly used model-free formalism could not be used to reflect the dynamic properties. The reduced spectral density function mapping approach was proven to be a useful tool to describe the overall and internal motion of molecules on the picosecond to nanosecond time-scale, and conformational exchanges on the microsecond to millisecond time-scale. The local motions in DNA-free Genesis showed strong temperature dependence and the backbone dynamics of each secondary structural element responds to the temperature change differently, while the Genesis-DNA complex showed more stability with changing the temperatures. Furthermore, each DNA contact sequence of Genesis showed distinct dynamic perturbation after Genesis binds to DNA.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

NMR structure and dynamics of recombinant wild type and mutated jerdostatin, a selective inhibitor of integrin α1β1

NMR analysis of four recombinant jerdostatin molecules was assessed to define the structural basis of two naturally occurring gain-of-function events: C-terminal dipeptide processing and mutation of the active residue K21 to arginine. Removal of the highly mobile and a bulky C-terminal dipeptide produced pronounced chemical shift changes in the sequentially unconnected but spatially nearby α(1)β(1) inhibitory loop. Analysis of chemical shift divergence and (15)N backbone relaxation dynamics indicated differences in motions in the picosecond to nanosecond time scale, and the higher T(2) rate of S25, S26, and H27 of rJerK21 point to a slowdown in the microsecond to millisecond motions of these residues when compared with rJerR21. The evidence presented in this article converges on the hypothesis that dynamic differences between the α(1)β(1) recognition loops of rJerR21 and rJerK21 may influence the thermodynamics of their receptor recognition and binding. A decrease in the μs-ms time scale may impair the binding affinity by reducing the rate of possible conformations that the rJerK21 can adopt in this time scale.     

Long-range structural and dynamical changes induced by cofactor binding in DNA methyltransferase M.HhaI

The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.     

Dynamically Driven Protein Allostery Exhibits Disparate Responses for Fast and Slow Motions

There is considerable interest in the dynamic aspect of allosteric action, and in a growing list of proteins allostery has been characterized as being mediated predominantly by a change in dynamics, not a transition in conformation. For considering conformational dynamics, a protein molecule can be simplified into a number of relatively rigid microdomains connected by joints, corresponding to, e.g., communities and edges from a community network analysis. Binding of an allosteric activator strengthens intermicrodomain coupling, thereby quenching fast (e.g., picosecond to nanosecond) local motions but initiating slow (e.g., microsecond to millisecond), cross-microdomain correlated motions that are potentially of functional importance. This scenario explains allosteric effects observed in many unrelated proteins.     

NMR (13)C-relaxation study of base and sugar dynamics in GCAA RNA hairpin tetraloop

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.     

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.     

NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.     

NMR spectroscopic characterization of a beta-(1,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)8-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, 15N relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.     

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.     

Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole*

Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by (15)N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2- sec -4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature dependence of the motional parameters.     

Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.     

Interesting structural and dynamical behaviors exhibited by the AF-6 PDZ domain upon Bcr peptide binding

PDZ (postsynaptic density-95, disks large, zonula occludens-1) domains are small, protein-protein interaction modules that have multiple binding surfaces for the docking of diverse molecules. These domains can propagate signals from ligand-binding site to distal regions of the structure through allosteric communication. Recent works have revealed that picosecond to nanosecond time scale dynamics play a potential role in propagating long-range signals within a protein. Comparison of AF-6 PDZ domain structures in free and complex forms shows a conformation rearrangement of distal surface 2, which is far from the peptide binding groove. The relaxation dispersion experiments detected that the free AF-6 PDZ domain was sampling multiple conformations; millisecond dynamics mapped a network for allostery signal transmission throughout the AF-6 PDZ domain in the weak saturation state, and intramolecular motions were observed in distal surface 1 when the protein was saturated. These results provide evidence that the allosteric process in the AF-6 PDZ domain is not two-state; instead, the millisecond dynamic network provides a mechanism for the transmission of allosteric signals throughout a protein. Interestingly, the two distal surfaces of the AF-6 PDZ domain respond differently to peptide binding; distal surface 1 changes in millisecond dynamics, whereas distal surface 2 undergoes structural rearrangement. The significance of the different response patterns in the signaling pathway and its relevance to the function of the AF-6 PDZ domain should be studied further.     

Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution

Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.     

Solution structure of human sorting nexin 22

The sorting nexins (SNXs) constitute a large group of PX domain-containing proteins that play critical roles in protein trafficking. We report here the solution structure of human sorting nexin 22 (SNX22). Although SNX22 has <30% sequence identity with any PX domain protein of known structure, it was found to contain the alpha/beta fold and compact structural core characteristic of PX domains. Analysis of the backbone dynamics of SNX22 by NMR relaxation measurements revealed that the two walls of the ligand binding cleft undergo internal motions: on the picosecond timescale for the beta1/beta2 loop and on the micro- to millisecond timescale for the loop between the polyproline motif and helix alpha2. Regions of the SNX22 structure that differ from those of other PX domains include the loop connecting strands beta1 and beta2 and the loop connecting helices alpha1 and alpha2, which appear to be more mobile than corresponding loops in other known structures. The interaction of dibutanoyl-phosphatidylinositol-3-phosphate (dibutanoyl-PtdIns(3)P) with SNX22 was investigated by an NMR titration experiment, which identified the binding site in a basic cleft and indicated that ligand binding leads only to a local structural rearrangement as has been found with other PX domains. Because motions in the loops are damped out when dibutanoyl-PtdIns(3)P binds, entropic effects could contribute to the lower affinity of SNX22 for this ligand compared to other PX domains.     

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.     

Hsc70-interacting HPD loop of the J domain of polyomavirus T antigens fluctuates in ps to ns and micros to ms

The backbone dynamics of the J domain from polyomavirus T antigens have been investigated using 15N NMR relaxation and molecular dynamics simulation. Model-free relaxation analysis revealed picosecond to nanosecond motions in the N terminus, the I-II loop, the C-terminal end of helix II through the HPD loop to the beginning of helix III, and the C-terminal end of helix III to the C terminus. The backbone dynamics of the HPD loop and termini are dominated by motions with moderately large amplitudes and correlation times of the order of a nanosecond or longer. Conformational exchange on the microsecond to millisecond timescale was identified in the HPD loop, the N and C termini, and the I-II loop. A 9.7ns MD trajectory manifested concerted swings of the HPD loop. Transitions between major and minor conformations of the HPD loop featured distinct patterns of change in backbone dihedral angles and hydrogen bonds. Fraying of the C-terminal end of helix II and the N-terminal end of helix III correlated with displacements of the HPD loop. Correlation of crankshaft motions of Gly46 and Gly47 with the collective motions of the HPD loop suggested an important role of the two glycine residues in the mobility of the loop. Fluctuations of the HPD loop correlated with relative reorientation of side-chains of Lys35 and Asp44 that interact with Hsc70.