Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly­(ethylene glycol) (PEG) can selectively shift the equilibrium to the “on” state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the “ordered off” state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE

Various plant diagnoses

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly­(ethylene glycol) (PEG) can selectively shift the equilibrium to the “on” state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the “ordered off” state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE

Expression, purification, crystallization and X-ray data collection for RAS and its mutants

This article expands on crystal structure data for human H-RAS with mutations at position Y137, briefly described in a paper on the effects of phosphorylation of Y137 by ABL kinases (Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding, published in the FASEB Journal [1]). The crystal structures of the Y137E mutant (phosphorylation mimic) and of the Y137F mutant (without the hydroxyl group where phosphorylation occurs) were deposited in the Protein Data Bank with PDB codes 4XVQ (H-RASY137E) and 4XVR (H-RASY137F). This article includes details for expression and purification of RAS and its mutants with no affinity tags, in vitro exchange of guanine nucleotides, protein crystallization, X-ray data collection and structure refinement. Keywords: RAS GTPase, Protein purification, Nucleotide exchange, X-ray crystal structure

A High-Resolution Crystal Structure of a Psychrohalophilic α–Carbonic Anhydrase from <i>Photobacterium profundum</i> Reveals a Unique Dimer Interface

<div><p>Bacterial α–carbonic anhydrases (α-CA) are zinc containing metalloenzymes that catalyze the rapid interconversion of CO₂ to bicarbonate and a proton. We report the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, <i>Photobacterium profundum</i>. Size exclusion chromatography of the purified <i>P</i>. <i>profundum</i> α–CA (PprCA) reveals that the protein is a heterogeneous mix of monomers and dimers. Furthermore, an “in-gel” carbonic anhydrase activity assay, also known as protonography, revealed two distinct bands corresponding to monomeric and dimeric forms of PprCA that are catalytically active. The crystal structure of PprCA was determined in its native form and reveals a highly conserved “knot-topology” that is characteristic of α–CA’s. Similar to other bacterial α–CA’s, PprCA also crystallized as a dimer. Furthermore, dimer interface analysis revealed the presence of a chloride ion (Cl^-) in the interface which is unique to PprCA and has not been observed in any other α–CA’s characterized so far. Molecular dynamics simulation and chloride ion occupancy analysis shows 100% occupancy for the Cl^- ion in the dimer interface. Zinc coordinating triple histidine residues, substrate binding hydrophobic patch residues, and the hydrophilic proton wire residues are highly conserved in PprCA and are identical to other well-studied α–CA’s.</p></div

Buhrman, Robert Alan

Also available as a printed booklet and from the Dean of Faculty website https://theuniversityfaculty.cornell.edu/Memorial Statement for Robert Alan Buhrman, who died in 2021. The memorial statements contained herein were prepared by the Office of the Dean of the University Faculty of Cornell University to honor its faculty for their service to the university

Cartoon representation of the PprCA active site and oligomerization status of PprCA.

<p>(<b>A</b>) The highly conserved active site residues of PprCA showing Zinc ion in the center of the active site (gray sphere). The hydrophobic (Val117, Val127, Leu181, Val190 and Trp192), the hydrophilic (Tyr17, Asn69, Gln74, Thr182 and Thr183), and zinc coordinating residues (His96, His98 and His115) are similar to other well characterized α–CA’s. (<b>B</b>) Active site water network involved in proton transfer is shown as red spheres. 2Fo-Fc electron density contoured at 8.0σ (green) and 1.0σ in blue. (<b>C</b>) Purified PprCA was analyzed on a Hiprep 16/60 Sephacryl S-200 size-exclusion column that was equilibrated with 20 mM Tris pH 7.5 and 150 mM NaCl. Peaks A and B correspond to dimer and monomer, respectively. The elution fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. A single ~35 kDa band was observed for both dimeric and monomeric PprCA. MW–Molecular-weight marker, lanes 43 to 51 (elution fractions representing PprCA dimer) and lanes 52 to 64 (elution fractions representing PprCA monomer). (<b>D</b>) Purified PprCA was separated under both reducing and non-reducing conditions on a SDS-PAGE. Lanes were loaded with PprCA from monomer fraction, dimer fraction and from a sample containing a mixture of monomers and dimers. The gels were stained either by Coomassie blue or subjected to protonography. Protonography was performed according to De Luca et al, 2015 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168022#pone.0168022.ref009" target="_blank">9</a>]. The gel was incubated in CO₂ enriched water for 5 to 15 seconds at room temperature. Appearance of distinctive yellow bands in gels subjected to protonography indicates both monomeric (~27 kDa) and dimeric (~58 kDa) PprCA is catalytically active.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p