The structural and dynamic basis for co-operative ligand binding in the KIX domain of CBP

Die KIX Domäne des Transkriptionscoaktivators CREB Binding Protein (CBP) bindet Transkriptionsfaktoren kooperativ. Die Bindungsaffinitäten der jeweiligen Bindungspartner der KIX Domäne werden allosterisch reguliert: das Binden eines Transkriptionsfaktors an einer der beiden Bindungsstellen der KIX Domäne führt zur erhöhten Affinität des zweiten Liganden an einer zweiten, räumlich entfernten Bindungsstelle. Unsere Relaxations-Dispersions Messungen mittels Kernresonanzspektroskopie zeigen, dass das Binden eines einzelnen Transkriptionsfaktors, der Aktivierungsdomäne von Mixed Lineage Leukemia (MLL) die Formierung einer zweiten, niedrig populierten Proteinkonformation, einem sogenannten angeregten Zustand induziert, dies ist in Übereinstimmung mit dem Konformations-Selektions Modell, welches die Ligandenerkennung eines Proteins beschreibt. Die Daten zeigen, dass dem hydrophoben Kern der KIX Domäne, welcher die beiden Bindungsstellen der Liganden verknüpft, eine entscheidende Rolle im Mechanismus des kooperativen Bindens zukommt. Wir konnten zeigen, dass die allosterische Informationsübertragung, zwischen den Bindungsstellen über diesen hydrophoben Kern geschieht. Weiters beschreiben wir die dreidimensionalen Proteinstrukturen des binären KIX Komplexes mit MLL und des ternären KIX Komplexes mit MLL und pKID in wässriger Lösung. Der hydrophobe Kern der KIX Domäne unterscheidet sich signifikant in den beiden Komplexen. Das Binden von pKID an den binären, aus KIX und MLL bestehenden Komplex führt zu einer definierten konformationellen Umlagerung im hydrophoben Kern welche den Pfad der allosterischen Informationsübertragung in der KIX Domäne konstituiert. Unsere Resultate liefern eine detaillierte strukturelle und dynamische Beschreibung der allosterischen Kommunikation in der KIX Domäne und Sie zeigen, dass Umlagerungen im hydrophoben Kern mechanistisch entscheidend sind.The KIX domain of the transcriptional co-activator CREB binding protein (CBP) cooperatively binds transcription factors. Allostery plays a significant role in the regulation of the affinities of the ligand molecules: transcription factor binding to one surface of the KIX domain enhances interactions with transcription factors to the second, remote binding site of the protein. Our nuclear magentic resonance spin relaxation studies reveals that binding of a single transcription factor molecule, the activation domain of mixed lineage leukemia (MLL) induces the formation of a low-populated (excited) conformer of KIX that resembles the conformation of the protein in the presence of the second ligand, in agreement with the conformational selection model of ligand recognition. The data highlights the mechanistic significance of the hydrophobic core of the KIX domain that bridges the two binding sites for the observed binding cooperativity revealing the allosteric pathway in KIX that connects the two binding sites. Furthermore, we describe the three-dimensional solution structures of the binary complex of KIX with MLL and the ternary complex of KIX formed by binding MLL and pKID to the KIX domain. Significant differences are found for the hydrophobic core of the KIX domain. Binding of pKID to the binary complex formed by KIX with MLL causes the hydrophobic core that constitutes the allosteric pathway to undergo a defined conformational transition, which propagates to the remote binding surface of the protein. Our results provide a structural rationalization of allosteric communication of the KIX domain, highlighting the mechanistic significance of the re-packing of the hydrophobic core of KIX

NMR Methods to Study Dynamic Allostery.

Nuclear magnetic resonance (NMR) spectroscopy provides a unique toolbox of experimental probes for studying dynamic processes on a wide range of timescales, ranging from picoseconds to milliseconds and beyond. Along with NMR hardware developments, recent methodological advancements have enabled the characterization of allosteric proteins at unprecedented detail, revealing intriguing aspects of allosteric mechanisms and increasing the proportion of the conformational ensemble that can be observed by experiment. Here, we present an overview of NMR spectroscopic methods for characterizing equilibrium fluctuations in free and bound states of allosteric proteins that have been most influential in the field. By combining NMR experimental approaches with molecular simulations, atomistic-level descriptions of the mechanisms by which allosteric phenomena take place are now within reach

Structural Biology Communications Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P i carrier SCaMC1

SCaMC is an ATP-Mg/P i carrier protein located at the mitochondrial inner membrane. SCaMC has an unusual N-terminal Ca 2+ -binding domain (NTD) in addition to its characteristic six-helix transmembrane bundle. The NTD of human SCaMC1 (residues 1-193) was expressed and purified in order to study its role in Ca 2+ -regulated ATP-Mg/P i transport mediated by its transmembrane domain. While Ca 2+ -bound NTD could be crystallized, the apo state resisted extensive crystallization trials. Selenomethionine-labeled Ca 2+ -bound NTD crystals, which belonged to space group P6 2 22 with one molecule per asymmetric unit, diffracted X-rays to 2.9 Å resolution

Dynamics from residual dipolar couplings (RDCs).

<p>(A) In isotropic solution, rotational diffusion averages dipolar couplings to zero and only scalar couplings <i>J</i> are observed. Weak molecular alignment of proteins impedes averaging of dipolar couplings to zero, and RDCs greater than or less than zero add to line splittings. (B) Residual dipolar couplings contain site-specific information on the orientation of internuclear vectors with respect to a molecular reference frame. Population-weighted averaged RDCs are observed if internal dynamics cause dipolar vectors to reorient. By combining experimental data from multiple molecular alignment media, structural and dynamic contributions can be separated to extract RDC-derived order parameters.</p

Relaxation dispersion experiments.

<p>(A) Transitions (exchange) between two states, A and B, causes line broadening of resonances in NMR spectra if the chemical shifts of the two states are different (Δω ≠ 0) and the exchange rate constant, <i>k</i>_ex, is in the micro- to millisecond time range. (B) In the typical experimental setup for CPMG relaxation dispersion measurements, resonance intensities at multiple protein sites (e.g., all backbone amide NH groups) are measured at variable CPMG frequencies (bottom). Relaxation dispersion profiles are obtained by converting these intensities to transverse relaxation rates (top). (C) Analysis of RD profiles yields information on kinetic (<i>k</i>_ex), thermodynamic (fractional populations <i>p</i>_A, <i>p</i>_B), and structural (Δω) parameters of the underlying dynamic exchange process(es). RD experiments provide this information only for protein sites with different local structures in states A and B (Δω ≠ 0).</p

Determination of NMR order parameters.

<p>(A) Processes in the pico- to nanosecond time regime can be probed by experiments that monitor the relaxation rates of different spin modes. Relaxation rates at multiple sites in a protein are determined from exponential fits of resonance intensities in a time series. (B) Analysis of the experimental data within the model-free approach separates nanosecond timescale contributions arising from rotational diffusion of the protein as a whole (τ_c) from (typically) picosecond contributions due to internal bond vector fluctuations, for which amplitudes (<i>S</i>²), timescale (τ_e), and, if applicable, information on additional motions are obtained.</p

Magnetization exchange experiments.

<p>(A) In cases in which separate resonances are observed for states A and B, transitions between these states occurring in approximately hundreds of milliseconds can be monitored by magnetization exchange. In these experiments, exchange cross-peaks (shown in red) are observed that are directly related to the interconversion between A and B. (B) Analysis of peak intensities in magnetization exchange spectra with variable delay periods yields kinetic (<i>k</i>_ex) and thermodynamic (<i>p</i>_A, <i>p</i>_B) information at multiple sites (e.g., NH groups) in proteins.</p

Three-dimensional structures of allosteric proteins.

<p>(A) The homodimeric catabolite activator protein (CAP) bound to two molecules of cAMP (green spheres; Protein Data Bank [PDB] identifier 1G6N) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004620#pcbi.1004620.ref011" target="_blank">11</a>]. (B) The KIX domain of CREB-binding protein (CBP; blue) in complex with the peptides mixed-lineage leukemia (MLL; top, dark green, residues 2,840−2,858) and phosphorylated kinase-inducible domain (pKID; light green, residues 116−149; PDB identifier 2LXT) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004620#pcbi.1004620.ref012" target="_blank">12</a>]. (C) The PBX1 homeodomain (PBX-HD, blue) bound to DNA (green) and the HoxB1 homeodomain peptide (light blue, residues 177−185; PDB identifier 1B72) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004620#pcbi.1004620.ref013" target="_blank">13</a>]. (D) The 20<i>S</i> core particle proteasome (20<i>S</i> CP); α- and β-subunits are shown in light and dark blue, respectively (PDB identifier 3C91) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004620#pcbi.1004620.ref014" target="_blank">14</a>]. (E) The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS), subunits HisH (light blue) and HisF (dark blue). The allosteric effector PRFAR (dark green spheres) and the substrate glutamine (light green spheres) are shown (PDB identifier 1OX5) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004620#pcbi.1004620.ref015" target="_blank">15</a>]. Prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.41, Schrödinger LLC).</p