An official website of the United States government
How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.
more »
« less
Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography
Abstract Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.
more »
« less
- Award ID(s):
- 1231306
- PAR ID:
- 10463648
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Chemistry
- Volume:
- 15
- Issue:
- 11
- ISSN:
- 1755-4330
- Format(s):
- Medium: X Size: p. 1549-1558
- Size(s):
- p. 1549-1558
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Proteins perform their biological functions through motion. Although high throughput prediction of the three-dimensional static structures of proteins has proved feasible using deep-learning-based methods, predicting the conformational motions remains a challenge. Purely data-driven machine learning methods encounter difficulty for addressing such motions because available laboratory data on conformational motions are still limited. In this work, we develop a method for generating protein allosteric motions by integrating physical energy landscape information into deep-learning-based methods. We show that local energetic frustration, which represents a quantification of the local features of the energy landscape governing protein allosteric dynamics, can be utilized to empower AlphaFold2 (AF2) to predict protein conformational motions. Starting from ground state static structures, this integrative method generates alternative structures as well as pathways of protein conformational motions, using a progressive enhancement of the energetic frustration features in the input multiple sequence alignment sequences. For a model protein adenylate kinase, we show that the generated conformational motions are consistent with available experimental and molecular dynamics simulation data. Applying the method to another two proteins KaiB and ribose-binding protein, which involve large-amplitude conformational changes, can also successfully generate the alternative conformations. We also show how to extract overall features of the AF2 energy landscape topography, which has been considered by many to be black box. Incorporating physical knowledge into deep-learning-based structure prediction algorithms provides a useful strategy to address the challenges of dynamic structure prediction of allosteric proteins.more » « less
-
Proteins are the workhorses of the cell. The shape that a protein molecule adopts enables it to carry out its role. However, a protein’s shape, or 'conformation', is not static. Instead, a protein can shift between different conformations. This is particularly true for enzymes – the proteins that catalyze chemical reactions. The region of an enzyme where the chemical reaction happens, known as the active site, often has to change its conformation to allow catalysis to proceed. Changes in temperature can also make a protein shift between alternative conformations. Understanding how a protein shifts between conformations gives insight into how it works. A common method for studying protein conformation is X-ray crystallography. This technique uses a beam of X-rays to figure out where the atoms of the protein are inside a crystal made of millions of copies of that protein. At room temperature or biological temperature, X-rays can rapidly damage the protein. Because of this, most crystal structures are determined at very low temperatures to minimize damage. But cooling to low temperatures changes the conformations that the protein adopts, and usually causes fewer conformations to be present. Keedy, Kenner, Warkentin, Woldeyes et al. have used X-ray crystallography from a very low temperature (-173°C or 100 K) to above room temperature (up to 27°C or 300 K) to explore the alternative conformations of an enzyme called cyclophilin A. These alternative conformations include those that have previously been linked to this enzyme’s activity. Starting at a low temperature, parts of the enzyme were seen to shift from having a single conformation to many conformations above a threshold temperature. Unexpectedly, different parts of the enzyme have different threshold temperatures, suggesting that there isn’t a single transition across the whole protein. Instead, it appears the way a protein’s conformation changes in response to temperature is more complex than was previously realized. This result suggests that conformations in different parts of a protein are coupled to each other in complex ways. Keedy, Kenner, Warkentin, Woldeyes et al. then performed X-ray crystallography at room temperature using an X-ray free-electron laser (XFEL). This technique can capture the protein’s structure before radiation damage occurs, and confirmed that the alternative conformations observed were not affected by radiation damage. The combination of X-ray crystallography at multiple temperatures, new analysis methods for identifying and measuring alternative conformations, and XFEL crystallography should help future studies to characterize conformational changes in other proteins.more » « less
-
Abstract The catalytic activity of human glutathione S‐transferase A1‐1 (hGSTA1‐1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C‐terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand‐free state of the hGSTA1‐1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand‐free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand‐free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds‐timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand‐free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1‐1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1‐1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare‐events sampling strategy to gain mechanistic information on protein function at the atomic level.more » « less
-
Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.more » « less
