Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
, 48 (2), 423-30

Keep on Moving: Discovering and Perturbing the Conformational Dynamics of Enzymes

Affiliations
Review

Keep on Moving: Discovering and Perturbing the Conformational Dynamics of Enzymes

Gira Bhabha et al. Acc Chem Res.

Abstract

CONSPECTUS: Because living organisms are in constant motion, the word "dynamics" can hold many meanings to biologists. Here we focus specifically on the conformational changes that occur in proteins and how studying these protein dynamics may provide insights into enzymatic catalysis. Advances in integrating techniques such as X-ray crystallography, nuclear magnetic resonance, and electron cryomicroscopy (cryo EM) allow us to model the dominant structures and exchange rates for many proteins and protein complexes. For proteins amenable to atomic resolution techniques, the major questions shift from simply describing the motions to discovering their role in function. Concurrently, there is an increasing need for using perturbations to test predictive models of dynamics-function relationships. Examples are the catalytic cycles of dihydrofolate reductase (DHFR) and cyclophilin A (CypA). In DHFR, mutations that alter the ability of the active site to sample productive higher energy states on the millisecond time scale reduce the rate of hydride transfer significantly. Recently identified rescue mutations restore function, but the mechanism by which they do so remains unclear. The exact role of any changes in the dynamics remains an open question. For CypA, a network of side chains that exchange between two conformations is critical for catalysis. Mutations that lock the network in one state also reduce catalytic activity. A further understanding of enzyme dynamics of well-studied enzymes such as dihydrofolate reductase and cyclophilin A will lead to improvement in ability to modulate the functions of computationally designed enzymes and large macromolecular machines. In designed enzymes, directed evolution experiments increase catalytic rates. Detailed X-ray studies suggest that these mutations likely limit the conformational space explored by residues in the active site. For proteins where atomic resolution information is currently inaccessible, other techniques such as cryo-EM and high-resolution single molecule microscopy continue to advance. Understanding the conformational dynamics of larger systems such as protein machines will likely become more accessible and provide new opportunities to rationally modulate protein function.

Figures

Figure 1
Figure 1
Protein dynamics occur at different time scales. (a) Motions on the picosecond to nanosecond scale involve small changes in backbone or side chain torsion angles. Calcium bound calmodulin (1exr, upper) exhibits conformational heterogeneity on the interface of the peptide binding site. The residual conformational entropy of binding, depends on side chains sampling alternative conformations as exemplified by Met36 and Leu39 (lower). Electron density contoured to 2.5 e3 in a dark blue mesh and 0.8 e3 in cyan volume representation. The lessons from calmodulin likely apply to enzymes where the loss of conformational entropy associated with the rigidification of active-site loops or side chains can specifically weaken binding to substrate or product complexes and promote flux through the catalytic cycle. (b) A model of ubiquitin (2k39) derived from RDC data reporting on motions up to microseconds is shown as cartoon, with the other models in the ensemble shown as transparent ribbons (upper). The dynamic β1β2 loop moves between alternative loop conformations, represented as sticks (upper and lower). The population of the up (cyan), mid (blue), and down (purple) β1β2 conformations can be a critical determinant of binding preferences for protein–protein interactions. The rates of transition between these states discriminate between induced fit and conformational selection mechanisms,, which can influence catalytic mechanisms and inhibitor discovery. (c) For enzymes, loop motions on the millisecond time scale are often rate limiting for catalytic cycles, with essential roles for governing ligand flux and repositioning key catalytic residues for catalysis. The WPD loop of protein tyrosine phosphatase 1B (PTP1B) moves between the “closed” (1sug, orange) and “open” (1t49, cyan) form on the millisecond time scale, forming the catalytically competent closed active site conformation. Further molecular detail of the two conformations are shown in the lower panel with electron density contoured to 0.3 e3. (d) The archaeal proteasome, a ∼700-kDa complex, controls active site access through the dynamic exchange of the N-terminus to block or reveal the central pore on the time scale of seconds. The structure of the proteasome is shown as a homoheptamer with each subunit in a different color (upper). In the lower panel, the ensemble of structures of the N-terminus of one of the seven subunits is shown in blue (2ku1). (e) Many enzymes enter long-lived states, with distinct catalytic activities, through stochastic fluctuations. Quaternary structure reconstruction of two RAS molecules (yellow) and son of sevenless (SOS, gray surface) complex. The Cdc25, REM, DH, histone, and PH domains of SOS are colored blue, green, orange, brown, and red, respectively (1xd2 (RAS) and 3ksy (SOS)). This complex exchanges between long-lived states with distinct catalytic rates. The structural basis of this exchange is currently unknown but likely involves rearrangements of protein–protein interfaces shown schematically in equilibrium. (f) The folded crystal structure (1ssx) of α-lytic protease (upper) is a kinetically trapped structure. After folding catalyzed by a proline domain, the kinetic barrier to unfolding makes this protein stable on the scale of years. The benzoyl moiety of Phe228 deviates by 6° from planarity. Removing this distortion can change the unfolding barrier from over a year to less than 2 weeks. Electron density contoured to 4.75 e3 (dark blue mesh, lower) and 0.5 e3 (cyan volume representation).
Figure 2
Figure 2
Dynamics in DHFR. (a) Crystal structures of E. coli DHFR show the Met20 loop in the occluded (1rx4, blue) and closed (1rx2, red) conformations. During the catalytic cycle of DHFR, this loop fluctuates between these conformations on the millisecond time scale. The ligands NADPH (left ligand) and folate (right ligand) are shown in orange and yellow, respectively. (b) Mutation of Asn23 to two proline residues (N23PP) shown as sticks in red and Ser148 to alanine (S148A) shown in pink reduce activity of ecDHFR. Mutation of Gly51 to the sequence PEKN (shown in blue) partially recovers the catalytic activity. The activity is increased further by the Leu28Phe (L28F, green) mutation. The structure of N23PP/PEKN (4gh8) is shown with NADPH shown in orange, with substrate mimic methotrexate in tan. (c) pH independent hydride transfer rates of different mutants show the quantitative effects of mutations that alter the dynamics of the Met20 loop and packing around the substrate.
Figure 3
Figure 3
Dynamics in CypA. (a) Exchanging residues detected by CPMG experiments show two groups with exchange rate kex = 1140 ± 200 s–1 (red) and kex = 2260 ± 200 s–1 (blue) in the absence of substrate. All residues with detectable dynamic exchange can be fit to one rate (∼2400 s–1) when the protein is saturated with substrate, which interconverts from cis to trans on the enzyme (1rmh). (b) Wild-type CypA (3k0n) shows two sets of conformations at room temperature. The network of side chains of residues S99, F113, M61, and R55 are shown with surface representations around sticks, with the major conformation in red and the minor conformation in orange. These residues lie across the central β strands shown in panel a. (c) The network of these four residues for the S99T mutant at room temperature only occupies the minor-like conformation, shown in green (3k0o).
Figure 4
Figure 4
Designed enzymes. (a) The Kemp eliminase reaction scheme. (b) Structure of designed Kemp eliminase prior to directed evolution (3nyd) with two alternative ligand conformations (cyan and purple). Electron density contoured to 1.5 e3 as a dark blue mesh, with a lower contour shown as a cyan volume representation at 0.3 e3. (c) Structure of final designed Kemp eliminase (4BS0) highlighting the hydrogen bond network. Disordered water replaces the acetate of the previous structure. Electron density contoured to 2.65 e3 as a dark blue mesh, with a lower contour shown as a cyan volume representation at 0.3 e3. Difference density contoured to 0.3 e3, colored green for positive and red for negative. Hydrogen bonds in hydrogen bond network drawn as dashed black lines. (d) Disorder of tryptophan residue 44 in final Kemp eliminase. The modeled alternative conformation deposited in the PDB is shown in dark green. Electron density contoured as in panel b. (e) Possible alternative conformation of catalytic Asp127 as detected by two positive difference peaks indicating alternative positions of the carboxylic oxygens that stabilize interactions with alternative water conformations. Electron density contoured as in panel c.
Figure 5
Figure 5
The challenge of larger protein machines. The motor domain of dynein, a microtubule-based motor protein belonging to the AAA family of enzymes, is shown, colored by domain (3VKH). The entire dynein heavy chain is considerably larger and is difficult to produce in quantities required for structural biology studies. For comparison, DHFR (1RX2) and CypA (2CPL) are shown to scale.

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

References

    1. Dror R. O.; Dirks R. M.; Grossman J. P.; Xu H.; Shaw D. E. Biomolecular simulation: A computational microscope for molecular biology. Annu. Rev. Biophys. 2012, 41, 429–452. - PubMed
    1. Henzler-Wildman K.; Kern D. Dynamic personalities of proteins. Nature 2007, 450, 964–972. - PubMed
    1. Ward A. B.; Sali A.; Wilson I. A. Biochemistry. Integrative structural biology. Science 2013, 339, 913–915. - PMC - PubMed
    1. Tuttle L. M.; Dyson H. J.; Wright P. E. Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle. Biochemistry 2013, 52, 3464–3477. - PMC - PubMed
    1. Fenwick R. B.; van den Bedem H.; Fraser J. S.; Wright P. E. Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E445–454. - PMC - PubMed

Publication types

Substances

Feedback