Dorothee Kern

Intrinsic dynamics of an enzyme underlies catalysis

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis–trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.

A hierarchy of timescales in protein dynamics is linked to enzyme catalysis

The synergy between structure and dynamics is essential to the function of biological macromolecules. Thermally driven dynamics on different timescales have been experimentally observed or simulated, and a direct link between micro- to milli-second domain motions and enzymatic function has been established. However, very little is understood about the connection of these functionally relevant, collective movements with local atomic fluctuations, which are much faster. Here we show that pico- to nano-second timescale atomic fluctuations in hinge regions of adenylate kinase facilitate the large-scale, slower lid motions that produce a catalytically competent state. The fast, local mobilities differ between a mesophilic and hyperthermophilic adenylate kinase, but are strikingly similar at temperatures at which enzymatic activity and free energy of folding are matched. The connection between different timescales and the corresponding amplitudes of motions in adenylate kinase and their linkage to catalytic function is likely to be a general characteristic of protein energy landscapes.

Intrinsic motions along an enzymatic reaction trajectory.

The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate–enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent ‘closed’ state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.

Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair

A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.

Hidden alternative structures of proline isomerase essential for catalysis.

A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

Antiparallel EmrE exports drugs by exchanging between asymmetric structures.

Small multidrug resistance transporters provide an ideal system to study the minimal requirements for active transport. EmrE is one such transporter in Escherichia coli. It exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy has surrounded the topology of the EmrE homodimer. Here we show that asymmetric antiparallel EmrE exchanges between inward- and outward-facing states that are identical except that they have opposite orientation in the membrane. We quantitatively measure the global conformational exchange between these two states for substrate-bound EmrE in bicelles using solution NMR dynamics experiments. Förster resonance energy transfer reveals that the monomers within each dimer are antiparallel, and paramagnetic relaxation enhancement NMR experiments demonstrate differential water accessibility of the two monomers within each dimer. Our experiments reveal a ‘dynamic symmetry’ that reconciles the asymmetric EmrE structure with the functional symmetry of residues in the active site.

Transient non-native hydrogen bonds promote activation of a signaling protein

Phosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.

Enzyme Dynamics During Catalysis Measured by NMR Spectroscopy

Many biological processes, in particular enzyme catalysis, occur in the microsecond to millisecond time regime. While the chemical events and static structural features of enzyme catalysis have been extensively studied, very little is known about dynamic processes of the enzyme during the catalytic cycle. Dynamic NMR methods such as ZZ-exchange, line-shape analysis, Carr-Purcell-Meiboom-Gill (CPMG), and rotating frame spin-lattice relaxation (R(1rho)) experiments are powerful in detecting conformational rearrangements with interconversion rates between 0.1 and 10(5) s(-1). In this chapter, the first application of these methods to enzymes during catalysis is described, in addition to studies on several other enzymes in their free states and in complex with ligands. From the experimental results of all systems, a picture arises in which flexibility in the microsecond to millisecond time regime is intrinsic and likely to be an essential property of the enzyme. Quantitative analysis of dynamics at multiple sites of the enzyme reveal large-scale collective motions. For several enzymes, the frequency of motion is comparable to the overall turnover rate, raising the possibility that conformational rearrangements may be rate limiting for catalysis in these enzymes.

Using ancient protein kinases to unravel a modern cancer drug’s mechanism

Evolution of dynamics affects function The drug Gleevac inhibits Abl kinases and is used to treat multiple cancers. The closely related Src kinases also play a role in cancer but are not inhibited effectively by Gleevac. Nevertheless, Gleevac-bound structures of Src and Abl are nearly identical. Based on this structural information and protein sequence data, Wilson et al. reconstructed the common ancestor of Src and Abl. Mutations that affected conformational dynamics caused Gleevac affinity to be gained on the evolutionary trajectory toward Abl and lost on the trajectory toward Src. Science, this issue p. 882 Characterization of ancestors of the kinases Src and Abl reveals why they respond differently to the cancer drug Gleevec. Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

Reassessment of the putative chaperone function of prolyl-cis/trans-isomerases

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non‐productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate‐limiting steps of folding. We reinvestigated, whether peptidyl‐propyl‐cis/trans‐isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.‐O et al. (1992) Science 258, 466–468; Rinfret, A. et al. (1994) Biochemistry 33, 1668–1673]. No increase in the yield of carbonic anhydrase‐II could be detected in the presence of three different propyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of propyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.