Renee Otten

Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

Natively unfolded linkers facilitate nuclear membrane protein import. Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.

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.

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria

Significance The tropomyosin-like protein, DivIVA, determines the site of growth and cell morphology in mycobacteria. Surprisingly, although DivIVA is located at the tip of the growing cell pole, cell wall addition is excluded from this site. Both late cell wall synthetic enzymes and new cell wall deposition occur at a subpolar space, distinct from the DivIVA-marked cell tip. Instead of directly recruiting terminal cell wall synthetic systems, DivIVA interacts with enzymes involved in the early steps of the cell wall precursor synthesis. These results suggest a unique organization of the polar elongasome, where cell wall precursors are concentrated at the cell tip by DivIVA and then incorporated into the nascent cell wall in an annular pattern at the subpolar zone. Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension.

Fluoride resistance and transport by riboswitch-controlled CLC antiporters

A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F-. We establish here that a set of randomly selected representatives from this “CLCF” clade protect Escherichia coli from F- toxicity, and that the purified proteins catalyze transport of F- in liposomes. Sequence alignments and membrane transport experiments using 19F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLCF proteins apart from all other known CLCs. First, CLCFs lack conserved residues that form the anion binding site in canonical CLCs. Second, CLCFs exhibit high anion selectivity for F- over Cl-. Third, at a residue thought to distinguish CLC channels and transporters, CLCFs bear a channel-like valine rather than a transporter-like glutamate, and yet are F-/H+ antiporters. Finally, F-/H+ exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1.

Comprehensive and Cost-Effective NMR Spectroscopy of Methyl Groups in Large Proteins

An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using [(1)H,(13)C]-D-glucose and approximately 100% D(2)O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable CHD(2) methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91% when taking into account that not all backbone assignments are available for this system. The remaining unassigned methyl groups belong to six leucine residues, caused by low cross-peak intensities, and four alanine residues due to degeneracy of the (13)C(alpha)/(13)C(beta) frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methyl-containing amino acids also for larger proteins.

Energetic dissection of Gleevec's selectivity toward human tyrosine kinases

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abls high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.

Probing Microsecond Time Scale Dynamics in Proteins by Methyl 1H Carr−Purcell−Meiboom−Gill Relaxation Dispersion NMR Measurements. Application to Activation of the Signaling Protein NtrCr

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ 1H Carr−Purcell−Meiboom−Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [1H, 13C]-d-glucose in ∼100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using 13C TOCSY NMR spectroscopy, as was recently demonstrated (Otten, R.; et al. J. Am. Chem. Soc.2010, 132, 2952−2960). In this Article, NMR pulse schemes are presented to measure 1H CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-δ1 and Thr-γ2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong 13C scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrCr, for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone 15N CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the 1H line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 ± 0.5) × 103 per second (i.e., τex = 64.7 ± 1.9 μs). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by 1H CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone 15N relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins.

The energy landscape of adenylate kinase during catalysis

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events: phosphoryl transfer (>105-fold) and lid opening (103-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.

Cavity as a Source of Conformational Fluctuation and High-Energy State: High-Pressure NMR Study of a Cavity-Enlarged Mutant of T4Lysozyme

Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the (1)H/(13)C heteronuclear single quantum correlation spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of >20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. (13)C and (1)H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state.

Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis

Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.