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.

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.

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.

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.

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.

Replacement of corn with mango meal for dairy goats / Sustitución de maíz por harina de mango para cabras lecheras / Substituição do milho pelo farelo de manga em cabras leiteiras

Summary Background: the high volume of mangos harvested during the season, associated with inappropriate management of the fruit after harvesting results in increased waste of this valuable resource. Mango fruit, rich in carbohydrates, has potential use in animal feeding. Objective: to evaluate the effect of replacing corn with whole mango meal (0, 330, 660, and 1000 g/Kg on dry matter basis) in the diets of dairy goats on rumen fermentation kinetics, intake, milk yield and composition, and economic results. Methods: eight lactating Saanen crossbred goats (48.7 ± 1.99 Kg body weight) were used in the experiment, which started 48 days postpartum and lasted until completing 124 days of lactation. The experimental design consisted of a double Latin square (4×4) with four treatments, four periods and four animals per square. Results: a reduction (p<0.05) was observed in gas production from total carbohydrates and fiber carbohydrates. Replacement of corn with whole mango meal showed no effect on DM intake (1,890 g/d), crude protein (278 g/d) and neutraldetergent fiber (959 g/d). Milk production (4% fat-corrected) and milk composition were not affected by the treatments, except for fat and myristoleic fatty acid contents. The economic evaluation showed a reduction in the total feeding cost and a better benefit:cost ratio. Conclusion: whole mango meal can replace corn up to 330 g/Kg in the diet of lactating goats.

Resumen Antecedentes: la alta producción de mango asociado con una gestión inadecuada durante la producción y pos-cosecha, resulta en un aumento de los residuos en el medio ambiente. Debido a que el mango es una fruta rica en carbohidratos (fuente de energía) es posible su utilización en la alimentación de los animales. Objetivo: evaluar el efecto de la sustitución de maíz con harina integral de mango (0, 330, 660 y 1000 g/Kg en base a materia seca) en las dietas de cabras lecheras sobre la cinética de fermentación ruminal, consumo, producción y composición láctea y evaluación económica de las dietas. Métodos: ocho cabras Saanen mestizas en lactación (48,7 ± 1,99 Kg de peso corporal) fueron utilizadas en el experimento desde el día 48 hasta el día 124 de lactancia. Se utilizó un diseño experimental doble cuadrado latino (4×4) con cuatro tratamientos, cuatro períodos y cuatro animales. Resultados: se observó una reducción (p<0,05) en la producción y volumen de gas de los carbohidratos totales y carbohidratos fibrosos. La sustitución de maíz con harina de mango integral no mostró ningún efecto sobre el consumo de materia seca (1890 g/d), proteína bruta (278 g/d) y fibra detergente neutra (959 g/d). La producción de leche (corregida al 4% de grasa) y su composición no fueron afectadas por los tratamientos, excepto para el contenido de grasa y ácido graso miristoleico. La evaluación económica mostró una reducción en el costo total de la alimentación y una mejor relación costo:beneficio. Conclusión: se recomienda sustituir el maíz por la harina de mango integral en hasta 330 g/Kg en las dietas de cabras lecheras.

Resumo Antecedentes: a elevada produção de manga associada a um manejo inadequado durante a produção e pós- colheita, resulta em aumento de resíduos no ambiente. Por ser uma fruta rica em carboidratos (fonte de energia) é possível a sua utilização na alimentação animal. Objetivo: avaliar o efeito da substituição do milho pelo farelo de manga integral (0, 330, 660 e 1000 g/Kg na matéria seca) na dieta de cabras leiteiras sobre a cinética de fermentação ruminal, consumo, produção e composição do leite, e também avaliação econômica das dietas. Métodos: oito cabras mestiças Saanen em lactação (48,7 ± 1,99 Kg de peso corporal) foram introduzidas no experimento 48 dias pós-parto e mantidas até 124 dias de lactação. Foi utilizado um delineamento em duplo quadrado Latino (4×4), com quatro tratamentos, quatro períodos e quatro animais por quadrado. Resultados: houve redução (p<0,05) na produção máxima de gás a partir dos carboidratos totais e o volume de gás produzido a partir de carboidratos fibrosos. A substituição do milho pelo farelo de manga integral não mostrou nenhum efeito sobre o consumo de matéria seca (1890 g/d), proteína bruta (278 g/d) e fibra em detergente neutro (959 g/d). A produção de leite corrigida para 4% de gordura e a composição do leite não foram afetadas pelos tratamentos, com exceção do teor de gordura e o ácido graxo miristoleico. A avaliação econômica mostrou uma redução no custo total da alimentação e uma melhor relação custo:benefício. Conclusão: recomenda-se substituir o milho pelo farelo de manga integral até 330 g/Kg na dieta de cabras leiteiras.

Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate.

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects.

The crystal structure of RAS was first solved 25 years ago. In spite of tremendous and sustained efforts, there are still no drugs in the clinic that directly target this major driver of human cancers. Recent success in the discovery of compounds that bind RAS and inhibit signaling has fueled renewed enthusiasm, and in-depth understanding of the structure and function of RAS has opened new avenues for direct targeting. To succeed, we must focus on the molecular details of the RAS structure and understand at a high-resolution level how the oncogenic mutants impair function. Structural networks of intramolecular communication between the RAS active site and membrane-interacting regions on the G-domain are disrupted in oncogenic mutants. Although conserved across the isoforms, these networks are near hot spots of protein-ligand interactions with amino acid composition that varies among RAS proteins. These differences could have an effect on stabilization of conformational states of interest in attenuating signaling through RAS. The development of strategies to target these novel sites will add a fresh direction in the quest to conquer RAS-driven cancers. Clin Cancer Res; 21(8); 1810-8. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD.

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

The Ras-Membrane Interface: Isoform-specific Differences in The Catalytic Domain.

The small GTPase Ras is mutated in about 20% of human cancers, primarily at active site amino acid residues G12, G13, and Q61. Thus, structural biology research has focused on the active site, impairment of GTP hydrolysis by oncogenic mutants, and characterization of protein-protein interactions in the effector lobe half of the protein. The C-terminal hypervariable region has increasingly gained attention due to its importance in H-Ras, N-Ras, and K-Ras differences in membrane association. A high-resolution molecular view of the Ras-membrane interaction involving the allosteric lobe of the catalytic domain has lagged behind, although evidence suggests that it contributes to isoform specificity. The allosteric lobe has recently gained interest for harboring potential sites for more selective targeting of this elusive "undruggable" protein. The present review reveals critical insight that isoform-specific differences appear prominently at these potentially targetable sites and integrates these differences with knowledge of Ras plasma membrane localization, with the intent to better understand the structure-function relationships needed to design isoform-specific Ras inhibitors.

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.