An introduction to NMR-based approaches for measuring protein dynamics

Kleckner, Ian R.; Foster, Mark P.

doi:10.1016/j.bbapap.2010.10.012

Cited by 471 publications

(526 citation statements)

References 202 publications

(270 reference statements)

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“…It is natural that allosteric regulation depends on modulation of protein motions, because substrate binding and catalysis are linked to conformational dynamics (19,20). Unlike static X-ray structures, solution NMR relaxation dispersion techniques have been instrumental in providing high-resolution conformational exchange information using sitespecific isotope labeling (21). In NMR studies of CypA, a dynamic continuum has been identified such that the relaxation profiles cannot be globally fit to one or two exchange phenomena and are instead indicative of more localized motions (22).…”

mentioning

confidence: 99%

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Doshi

Holliday

Eisenmesser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

152

186

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Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.allostery | enzyme dynamics | residue-residue contacts | Cyclophilin A | molecular dynamics

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mentioning

confidence: 99%

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Doshi

Holliday

Eisenmesser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

152

186

View full text Add to dashboard Cite

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“…1, E and F) and/or by partially stabilizing additional states, distinct from the fully active or fully inactive states, in which the activation function of the CBD is compromised. To test these hypotheses, here we comparatively analyze the S672R and WT HCN4 CBDs in both apo-and holo-forms using NMR spectroscopy, which reports on dynamics at residue resolution and over multiple time scales (30,39). Furthermore, we examined the S672R versus WT relaxation and chemical shift changes, which are exquisitely sensitive atomic reporters of subtle but functionally relevant allosteric perturbations that are often elusive to other structural techniques (27,30).…”

mentioning

confidence: 99%

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

Boulton¹,

Akimoto

Akbarizadeh

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo-and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

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“…More importantly, residues in the phosphoacceptor region, as well as helices H0 and H5, showed substantially greater intensity changes yet no chemical shift perturbations. This suggests that these residues undergo pronounced exchange broadening 30 owing either to direct contacts with the kinase, as would be expected for the phosphoacceptors, or to indirect conformational perturbations. Indeed, when mapped on a homology model of PntP2 142-252 , the residues in helices H0 and H5 are both in close proximity and adjacent to side chains in the PNT domain shown previously by mutagenesis to be involved in docking interactions with the ERK2 Rolled [ Fig.…”

Section: Map Kinase Docking By Pointed-p2mentioning

confidence: 96%

“…However, it is difficult to estimate the nature of the conformations sampled by these fast motions from 15 N relaxation measurements alone. 30 More importantly, rapid amide HX was detected for residues throughout helices H0/H1 [ Fig. 4(A)].…”

Section: Pointed-p2 Pnt Domain Contains a Dynamic Helix H0mentioning

confidence: 99%

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

2012

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Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, 15 N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site.

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An introduction to NMR-based approaches for measuring protein dynamics

Cited by 471 publications

References 202 publications

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

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