Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351–357] were applied to the 9- cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 s, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271–284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.     

Analysis of the internal motion of free and ligand-bound human lysozyme by use of 15N NMR relaxation measurement: a comparison with those of hen lysozyme

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.     

Internal dynamics of d(CGCAAATTTGCG)2: a comparison of NMR relaxation measurements with a molecular dynamics simulation

We report the analysis of a 250 ps molecular dynamics simulation of the dodecamer d(CGCAAATTT-GCG)₂ immersed in a rectangular box of 3469 water molecules with 22 Na⁺ counterions. The internal dynamics of the molecule were investigated by studying the relevant autocorrelation functions related to the ¹³C-NMR relaxation parameters of the C1′-H1′ bonds of the sugar rings. The calculated effective correlation times τ _e (∼13 ps) and the order parameter S² (∼0.82) of the Lipari and Szabo formalism (Lipari and Szabo 1982a, b) are in satisfactory agreement with those determined previously by NMR (Gaudin et al. 1995, 1996). ¹H-¹H NOE buildups have also been measured experimentally and agree with those computed from the simulation. These results validate the simulation, and a more detailed analysis of the internal dynamics of the dodecamer was undertaken. Analysis of the distributions and of the autocorrelation functions of the glycosidic angle flucuations χ shows that the rotational motion of the sugar rings about their glycosidic bond conforms to a restricted diffusion mechanism. The amplitude of the motions and the diffusion constant are 20° and 17.10⁹ rad²s^–1 respectively. These values are in good agreement with ¹³C NMR data. Furthermore the simulation allows us to rule out another model also consistent with the experiment, consisting of a two-state jump between a syn and an anti conformation. Received: 19 November 1996 / Accepted: 17 March 1997     

Structural and dynamic differences of the estrogen receptor DNA-binding domain, binding as a dimer and as a monomer to DNA: molecular dynamics simulation studies

Molecular dynamics (MD) simulations of the estrogen receptor DNA-binding domain (ERDBD) as a dimer in complex with its DNA response element (ERE) show a significant difference in both structure and dynamics, compared to a MD simulation of monomeric ERDBD bound to its half-site response element (EREH). The C-terminal zinc binding domain (Zn_II), including a region (helix II) which is in a helical conformation in ERE-(ERDBD)₂, is considerably more flexible in EREH-ERDBD than in the dimeric complex. In EREH-ERDBD, all helical hydrogen bonds in helix II are broken and the entire Zn_II region is detached from a hydrogen bonding network that in ERE-(ERDBD)₂ connects to other parts of the protein as well as to the DNA. The regions that become flexible in EREH-ERDBD are identical to the regions where the NMR solution structure of free ERDBD is poorly ordered. This strongly suggests that dimerisation of ERDBD is required for ordering of the Zn_II region and that monomeric binding to DNA is not sufficient for the ordering. This contrasts to the glucocorticoid receptor DNA-binding domain (GRDBD) which has essentially the same mobility (uniform and limited), regardless of whether it is free as a monomer in solution, bound as a monomer to its half-site response element or in a dimeric complex with the full response element. The hydrogen bonding network that connects Zn_II with other parts of the protein and to DNA is almost identical in ERDBD and GRDBD. However, in GRDBD there is also a serine (in the N-terminal zinc coordinating region) with a central role in this network, connecting to the Zn_II region. This serine is replaced by a glycine in ERDBD and we suggest that this substitution is sufficient for destabilisation of the network, thus leading to a more flexible Zn_II region, which becomes ordered first upon forming a complex with another ERDBD and DNA. Received: 6 March 1998 / Revised version: 22 June 1998 / Accepted: 2 September 1998     

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.     

Compensatory and long-range changes in picosecond-nanosecond main-chain dynamics upon complex formation: 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S², show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S² on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.