Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

Abstract

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.DOI: http://dx.doi.org/10.7554/eLife.07574.001

New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

“…We have addressed the challenge of discovering new allosteric sites and small-molecule inhibitors for PTP1B by taking advantage of two new techniques in X-ray crystallography that reveal minor conformational states of protein and ligands. First, multitemperature crystallography [12] can reveal previously hidden alternative conformations that enable biological functions. Here we use this approach in PTP1B to reveal coupled conformations that colocalize in a concerted allosteric network.…”

“…Our analysis indicates a complex, inter-connected network involving multiple aromatic stacking, hydrogen-bonding, van der Waals, and electrostatic interactions. To complement this model-based assessment with a map-based approach, for several residues in the pocket we quantified electron density as a function of temperature for atom positions that are unique to the minor conformation (i.e., do not overlap with any atoms in the major conformation), reasoning that residues which respond to temperature similarly may be energetically coupled [12]. The population of each minor conformation increases non-linearly with temperature ( Figure 4A) in a similar fashion as the open state of the WPD loop ( Figure 1D) and the disordered state of the α 7 helix (Figure 2A), in support of the idea that these various regions of the protein are mutually conformationally coupled.…”

Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

“…[1] Although loop closures directly over the active site have long been implicated in catalysis, [2] the importance of a highly tuned conformational landscape in optimizing active site chemistry is also increasingly apparent. [3] Biophysical probes, aided by computational work, are showing progress in identifying functionally relevant motions; [1a,4] however, our ability to design experimental methods that can distinguish the impact of global and local protein motions on isolated chemical steps remains a challenging and compelling issue. [5] Soybean lipoxygenase-1 (SLO-1, Figure 1), a prototype for the study of enzymatic C-H activation via hydrogen-tunneling, is providing a unique window into the subtle influence of protein motions on catalysis.…”

“…[6,7] In the present study, we focus on understanding the underlying interaction between two distinct classes of catalysis-linked protein motions in H-transfer reactions: local distance sampling that is dependent on substrate labeling with isotopes and global conformational landscapes that are independent of this labeling. [3,5] We systematically explore the combined impact of temperature and pressure on the full set of kinetics parameters for WT SLO-1 and a range of mutants with established kinetic properties at ambient pressure. Surprisingly, pressure is found to primarily influence the isotope-independent motions in SLO-1, leaving local, isotope-dependent motions virtually unperturbed.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…One of the simplest approaches to mounting a large number of crystals is the use of traditional loops, 17,184 micromounts, 185 or capillaries 16 to mount a slurry of crystals. 169 It is also possible to mount crystals grown directly on the mount, as in the case of the X-CHIP, where pinned droplets are protected against dehydration by a thin coating of oil (Figure 8(c)) 186,187 or the aforementioned graphene-based microfluidic devices.…”