Integrative, dynamic structural biology at atomic resolution—it's about time

Bedem, Henry van den; Fraser, James S.

doi:10.1038/nmeth.3324

Cited by 236 publications

(176 citation statements)

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“…Technological advances in structural biology have permitted insights (3)(4)(5) into how changes in protein structure and flexibility contribute to allostery (6)(7)(8)(9). Allostery likely employs a continuum of mechanisms, from domain or subunit rearrangements to predominantly side-chain and backbone dynamics (6-8, 10, 11), to affect biological regulation (1).…”

mentioning

confidence: 99%

Entropy redistribution controls allostery in a metalloregulatory protein

Capdevila

Braymer

Edmonds

et al. 2017

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

143

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Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Here we show that perturbations in equilibrium picosecond-nanosecond motions impact zinc (Zn)-induced allosteric inhibition of DNA binding by the Zn efflux repressor CzrA (chromosomal zinc-regulated repressor). DNA binding leads to an unanticipated increase in methyl side-chain flexibility and thus stabilizes the complex entropically; Zn binding redistributes these motions, inhibiting formation of the DNA complex by restricting coupled fast motions and concerted slower motions. Allosterically impaired CzrA mutants are characterized by distinct nonnative fast internal dynamics "fingerprints" upon Zn binding, and DNA binding is weakly regulated. We demonstrate the predictive power of the wild-type dynamics fingerprint to identify key residues in dynamics-driven allostery. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities in a compact molecular scaffold. Technological advances in structural biology have permitted insights (3-5) into how changes in protein structure and flexibility contribute to allostery (6-9). Allostery likely employs a continuum of mechanisms, from domain or subunit rearrangements to predominantly side-chain and backbone dynamics (6-8, 10, 11), to affect biological regulation (1). Although these motions clearly impact site-site communication via defined molecular pathways (9) or energy level perturbations at distant sites (12), an allosteric effect without conformational change remains largely a theoretical postulate (10,13,14). In this context, changes in dynamics upon ligand binding (8,(15)(16)(17)(18)(19)(20) have long been predicted to impact allostery (5, 14, 17, 21); however, obtaining a quantitative experimental demonstration of the role of conformational entropy in allosteric systems remains challenging. Here we test these ideas in the context of heterotropic linkage and pinpoint fast internal dynamics as a primary contributor to functional, structure-encoded dynamics. We report an example of allostery where side-chain rotamer degeneracy is largely responsible for coupling two ligand-binding events through perturbations in a dynamic network that is required for both entropic and enthalpic driving forces.Our model system for studying heterotropic allostery is the transcriptional regulator CzrA (chromosomal zinc-regulated repressor) from the bacterial pathogen Staphylococcus aureus (22-25) ( Fig. 1 and Fig. S1A). Zinc homeostasis is critical to the virulence of S. aureus (26) and of many other microbial pathogens, and allows the organism to adapt to host-imposed zinc toxicity or limitation (27, 28). CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of metalloregulatory proteins (25, 29), individual members of which are capable of sensing a wide array of metal, metalloid, and nonmetal inducers on distinct sites on a relatively simple, homodimeric wi...

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mentioning

confidence: 99%

Entropy redistribution controls allostery in a metalloregulatory protein

Capdevila

Braymer

Edmonds

et al. 2017

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

143

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“…protein dynamics | normal modes | structural biology | diffuse scattering | liquid-like motions X -ray crystallography can be a key tool for elucidating the structural basis of protein motions that play critical roles in enzymatic reactions, protein-protein interactions, and signaling cascades (1). X-ray diffraction yields an ensemble-averaged picture of the protein structure: each photon simultaneously probes multiple unit cells that can vary because of internal rearrangements or changes to the crystal lattice.…”

mentioning

confidence: 99%

Measuring and modeling diffuse scattering in protein X-ray crystallography

Benschoten

Liu

González

et al. 2016

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

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X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.protein dynamics | normal modes | structural biology | diffuse scattering | liquid-like motions X -ray crystallography can be a key tool for elucidating the structural basis of protein motions that play critical roles in enzymatic reactions, protein-protein interactions, and signaling cascades (1). X-ray diffraction yields an ensemble-averaged picture of the protein structure: each photon simultaneously probes multiple unit cells that can vary because of internal rearrangements or changes to the crystal lattice. Bragg analysis of X-ray diffraction only yields the mean charge density of the unit cell, however, which fundamentally limits the information that can be obtained about protein dynamics (2, 3).An inherent limitation in Bragg analysis is that models with different concerted motions can yield the same mean charge density (4). The traditional approach assumes a single structural model with individual atomic displacement parameters (B factors). Given enough data, anisotropic displacement parameters can be modeled. When the data are more limited, translation-libration-screw (TLS) refinement, which models rigid-body motions of subdomains (5), is often used [22% of Protein Data Bank (PDB) depositions (6, 7)]. Variations in the TLS domains can predict very different motions that agree equally well with the Bragg data (8, 9).Bragg analysis can be combined ...

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“…Several approaches being currently developed for ensemble modeling find the minimal perturbations to a predefined, physically reasonable ensemble model that are necessary to recover experimental observables [see, e.g. (Beauchamp et al 2014;Pitera & Chodera, 2012;Stelzer et al 2011;van den Bedem & Fraser, 2015) and references therein]. REEFFIT is the first such approach developed for M 2 data .…”

Section: Multiple States Of Rna As a Major Challengementioning

confidence: 99%

RNA structure through multidimensional chemical mapping

Tian

Das

2016

Quart. Rev. Biophys.

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Abstract. The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M 2 ), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving the in vivo 'RNA structurome'.

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Integrative, dynamic structural biology at atomic resolution—it's about time

Cited by 236 publications

References 113 publications

Entropy redistribution controls allostery in a metalloregulatory protein

Entropy redistribution controls allostery in a metalloregulatory protein

Measuring and modeling diffuse scattering in protein X-ray crystallography

RNA structure through multidimensional chemical mapping

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