Allosteric site variants affect GTP hydrolysis on Ras.

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas.

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.

ABORDAGEM MULTIPROFISIONAL QUANTO AO USO E ABUSO DE DROGAS DURANTE A GESTAÇÃO: Usuárias do CAPS AD III / MULTIPROFESSIONALIONAL APPROACH TO DRUG USE AND ABUSE DURING PREGNANCY: Users of CAPS AD III

O artigo possui o objetivo de problematizar a abordagem multiprofissional no CAPS ADIII da Cidade de Vitória da Conquista, para gestantes usuárias de drogas dessa unidade. Isso porque, as drogas são substâncias que alteram as funções cerebrais e mudam a percepção, humor e comportamentos. Tal elemento utilizado na gestação ocasiona ao feto, morbidades e/ou aborto. Para evitar essas consequências, médicos ou enfermeiros da UBS devem-se, nas primeiras consultas de pré-natal, rastrear as gestantes dependentes das psicotrópicas, objetivando encaminhá-las ao CAPS ADIII. O estudo foi de caráter descritivo, exploratório, qualitativo de corte transversal. A coleta de dados foi por meio da aplicação de uma entrevista, utilizando a TALP. A pesquisa resultou em uma contribuição da compreensão de como é a abordagem e papel da equipe multiprofissional do CAPS ADIII, apontando às dificuldades e desafios enfrentados pela equipe na prática de suas atividades, na tentativa de incorporar não somente os cuidados físicos, mas também à abordagem psicológica e social. Considera-se a construção de uma diretriz específica para o tratamento das gestantes usuárias de drogas, uma vez que isso visa proporcionar uma melhor e eficaz articulação entre os CAPS ADIII e as UBS.

The article aims to problematic the multi-professional approach in CAPS ADIII of the city of Victory of Conquest, for pregnant women who use drugs from this unit. That's because, drugs are substances that alter brain functions and change perception, mood and behaviors. This element used in pregnancy causes the fetus, morbidity is and/or abortion. To avoid these consequences, UBS doctors or nurses should, at first consultations, examine pregnant women dependent on psychotropic women, with the aim of referring them to CAPS ADIII. The study was descriptive, exploratory, qualitative transverse. The data collection was through the application of an interview, using TALP. The research resulted in a contribution of understanding how is the approach and role of the multidisciplinary team of CAPS ADIII, pointing to the difficulties and challenges faced by the team in the practice of their activities, in an attempt to incorporate not only physical care, but also the psychological and social approach. It is considered the construction of a specific guideline for the treatment of pregnant drug users, since this aims to provide a better and effective articulation between caps ADIII and ubs.

Computational studies of the principle of dynamic-change-driven protein interactions.

Dynamic allostery emphasizes a role of entropy change manifested as a sole change in protein fluctuations without structural changes. This kind of entropy-driven effect remains largely understudied. The most significant examples involve protein-ligand interactions, leaving protein-protein interactions, which are critical in signaling and other cellular events, largely unexplored. Here we study an example of how protein-protein interaction (binding of Ras to the Ras binding domain [RBD] of the effector protein Raf) affects a subsequent protein association process (Ras dimerization) by quenching Ras internal motions through dynamic allostery. We also investigate the influence of point mutations or ambient temperature, respectively, on the protein dynamics and interaction of two other systems: in adenylate kinase (ADK) and in the EphA2 SAM:Ship2 SAM complex. Based on these examples, we postulate that there are different ways in which dynamic-change-driven protein interactions are manifested and that it is likely a general biological phenomenon.

Regulation of GTPase function by autophosphorylation.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Crystal Structure Reveals the Full Ras-Raf Interface and Advances Mechanistic Understanding of Raf Activation.

Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas-CRaf-RBD_CRD complex showing the Ras-Raf interface as a continuous surface on Ras, as seen in the KRas-CRaf-RBD_CRD structure. In molecular dynamics simulations of a Ras dimer model formed through the α4-α5 interface, the CRD is dynamic and located between the two Ras protomers, poised for direct or allosteric modulation of functionally relevant regions of Ras and Raf. We propose a molecular model in which Ras binding is involved in the release of Raf autoinhibition while the Ras-Raf complex dimerizes to promote a platform for signal amplification, with Raf-CRD centrally located to impact regulation and function.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor.

The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.

Diets containing cunhã (Clitoria ternatea L.) hay and forage cactus (Opuntia sp.) meal on production and meat quality of Boer crossbred goat.

The aim of this study was to evaluate the effect of association of forage cactus meal (CM) and cunhã hay (CH) on carcass traits, meat quality, and fatty acid (FA) profile of goats. Twenty-four goats were finished on diets composed with the association of CM and CH (83% CH + 17% CM, 67% CH + 33% CM, or 50% CH + 50% CM), corresponding to 60% of the total diet; the remainder was composed of 10% elephant grass and 30% concentrate. The control treatment consisted of 70% elephant grass and 30% concentrate. Carcass yield and commercial cut weight were higher in the animals fed CH and CM at any concentration (P < 0.05). Animals fed with intermediate and high CM content resulted in meat with better proportions of unsaturated FA (P = 0.0281), desirable FA (P = 0.0024), omega 6/omega 3 ratio (P = 0.0340), and hypo-/hypercholesterolemic index (P = 0.0140). The inclusion of CH and CM provided carcass yield and FA profile more favorable for consumer market and human health.

DRoP: Automated detection of conserved solvent-binding sites on proteins.

Water and ligand binding play critical roles in the structure and function of proteins, yet their binding sites and significance are difficult to predict a priori. Multiple solvent crystal structures (MSCS) is a method where several X-ray crystal structures are solved, each in a unique solvent environment, with organic molecules that serve as probes of the protein surface for sites evolved to bind ligands, while the first hydration shell is essentially maintained. When superimposed, these structures contain a vast amount of information regarding hot spots of protein-protein or protein-ligand interactions, as well as conserved water-binding sites retained with the change in solvent properties. Optimized mining of this information requires reliable structural data and a consistent, objective analysis tool. Detection of related solvent positions (DRoP) was developed to automatically organize and rank the water or small organic molecule binding sites within a given set of structures. It is a flexible tool that can also be used in conserved water analysis given multiple structures of any protein independent of the MSCS method. The DRoP output is an HTML format list of the solvent sites ordered by conservation rank in its population within the set of structures, along with renumbered and recolored PDB files for visualization and facile analysis. Here, we present a previously unpublished set of MSCS structures of bovine pancreatic ribonuclease A (RNase A) and use it together with published structures to illustrate the capabilities of DRoP.

Water in Ras Superfamily Evolution.

The Ras GTPase superfamily of proteins coordinates a diverse set of cellular outcomes, including cell morphology, vesicle transport, and cell proliferation. Primary amino acid sequence analysis has identified Specificity determinant positions (SDPs) that drive diversified functions specific to the Ras, Rho, Rab, and Arf subfamilies (Rojas et al. 2012, J Cell Biol 196:189-201). The inclusion of water molecules in structural and functional adaptation is likely to be a major response to the selection pressures that drive evolution, yet hydration patterns are not included in phylogenetic analysis. This article shows that conserved crystallographic water molecules coevolved with SDP residues in the differentiation of proteins within the Ras superfamily of small GTPases. The patterns of water conservation between protein subfamilies parallel those of sequence-based evolutionary trees. Thus, hydration patterns have the potential to help elucidate functional significance in the evolution of amino acid residues observed in phylogenetic analysis of homologous proteins. © 2019 Wiley Periodicals, Inc.

The Structural Basis of the Farnesylated and Methylated KRas4B Interaction with Calmodulin.

Ca2+-calmodulin (CaM) extracts KRas4B from the plasma membrane, suggesting that KRas4B/CaM interaction plays a role in regulating Ras signaling. To gain mechanistic insight, we provide a computational model, supported by experimental structural data, of farnesylated/methylated KRas4B1-185 interacting with CaM in solution and at anionic membranes including signaling lipids. Due to multiple interaction modes, we observe diverse conformational ensembles of the KRas4B-CaM complex. A highly populated conformation reveals the catalytic domain interacting with the N-lobe and the hypervariable region (HVR) wrapping around the linker with the farnesyl docking to the extended CaM's C-lobe pocket. Alternatively, KRas4B can interact with collapsed CaM with the farnesyl penetrating CaM's center. At anionic membranes, CaM interacts with the catalytic domain with large fluctuations, drawing the HVR. Signaling lipids establishing strong salt bridges with CaM prevent membrane departure. Membrane-interacting KRas4B-CaM complex can productively recruit phosphatidylinositol 3-kinase α (PI3Kα) to the plasma membrane, serving as a coagent in activating PI3Kα/Akt signaling.

Isoform-Specific Destabilization of the Active Site Reveals a Molecular Mechanism of Intrinsic Activation of KRas G13D.

Ras GTPases are mutated at codons 12, 13, and 61, with different frequencies in KRas, HRas, and NRas and in a cancer-specific manner. The G13D mutant appears in 25% of KRas-driven colorectal cancers, while observed only rarely in HRas or NRas. Structures of Ras G13D in the three isoforms show an open active site, with adjustments to the D13 backbone torsion angles and with disconnected switch regions. KRas G13D has unique features that destabilize the nucleotide-binding pocket. In KRas G13D bound to GDP, A59 is placed in the Mg2+ binding site, as in the HRas-SOS complex. Structure and biochemistry are consistent with an intermediate level of KRas G13D bound to GTP, relative to wild-type and KRas G12D, observed in genetically engineered mouse models. The results explain in part the elevated frequency of the G13D mutant in KRas over the other isoforms of Ras.

Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras.

Neutron protein crystallography (NPC) reveals the three-dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X-ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in enzyme catalysis, NPC has the advantage over X-ray crystallography that the crystals do not suffer radiation damage. The lack of radiation damage can be exploited to conduct in crystallo parametric studies. Here, the use of a single crystal of the small GTPase Ras to collect three neutron data sets at pD 8.4, 9.0 and 9.4 is reported, enabling an in crystallo titration study using NPC. In addition to revealing the behavior of titratable groups in the active site, the data sets will allow the analysis of allosteric water-mediated communication networks across the molecule, particularly regarding Cys118 and three tyrosine residues central to these networks, Tyr32, Tyr96 and Tyr137, with pKa values expected to be in the range sampled in our experiments.

Predicting X-ray solution scattering from flexible macromolecules.

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the structure and dynamics of a protein. Solution scattering from a rigid protein can be predicted from atomic coordinate sets to within experimental error. However, structural fluctuations of proteins in solution can lead to significant changes in the observed intensities. The magnitude and form of these changes contain information about the nature and spatial extent of structural fluctuations in the protein. Molecular dynamics (MD) simulations based on a crystal structure and selected force field generate models for protein internal motions, and here we demonstrate that they can be used to predict the impact of structural fluctuations on solution scattering data. In cases where the observed and calculated intensities correspond, we can conclude that the X-ray scattering provides direct experimental validation of the structural and MD results. In cases where calculated and observed intensities are at odds, the inconsistencies can be used to determine the origins of these discrepancies. They may be because of overestimates or underestimates of structural fluctuations in MD simulations, under-sampling of the structural ensemble in the simulations, errors in the structural model, or a mismatch between the experimental conditions and the parameters used in carrying out the MD simulation.

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D.

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH2p, with K-Ras in the state 1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state 1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies.

The K-Ras, N-Ras, and H-Ras Isoforms: Unique Conformational Preferences and Implications for Targeting Oncogenic Mutants.

Ras controls a multitude of cellular signaling processes, including cell proliferation, differentiation, and apoptosis. Deregulation of Ras cycling often promotes tumorigenesis and various other developmental disorders, termed RASopothies. Although the structure of Ras has been known for many decades, it is still one of the most highly sought-after drug targets today, and is often referred to as "undruggable." At the center of this paradoxical protein is a lack of understanding of fundamental differences in the G domains between the highly similar Ras isoforms and common oncogenic mutations, despite the immense wealth of knowledge accumulated about this protein to date. A shift in the field during the past few years toward a high-resolution understanding of the structure confirms the hypothesis that each isoform and oncogenic mutation must be considered individually, and that not all Ras mutations are created equal. For the first time in Ras history, we have the ability to directly compare the structures of each wild-type isoform to construct a "base-line" understanding, which can then be used as a springboard for analyzing the effects of oncogenic mutations on the structure-function relationship in Ras. This is a fundamental and large step toward the goal of developing personalized therapies for patients with Ras-driven cancers and diseases.

Neutron scattering in the biological sciences: progress and prospects.

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.

An engineered protein antagonist of K-Ras/B-Raf interaction.

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.

The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects.

H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.