The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Gorfe, Alemayehu A.

doi:10.1371/journal.pcbi.1002394

Cited by 50 publications

(53 citation statements)

References 79 publications

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“…Prior studies on allostery have primarily focused on the structure and dynamics of the protein itself; whereas, a few studies highlight the effect of solvation forces and water-mediated interactions toward allosteric modulation (49,50). Fig.…”

Section: Resultsmentioning

confidence: 99%

Hidden electrostatic basis of dynamic allostery in a PDZ domain

Kumawat

Chakrabarty

2017

Proc. Natl. Acad. Sci. U.S.A.

138

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Allosteric effect implies ligand binding at one site leading to structural and/or dynamical changes at a distant site. PDZ domains are classic examples of dynamic allostery without conformational changes, where distal side-chain dynamics is modulated on ligand binding and the origin has been attributed to entropic effects. In this work, we unearth the energetic basis of the observed dynamic allostery in a PDZ3 domain protein using molecular dynamics simulations. We demonstrate that electrostatic interaction provides a highly sensitive yardstick to probe the allosteric modulation in contrast to the traditionally used structure-based parameters. There is a significant population shift in the hydrogen-bonded network and salt bridges involving side chains on ligand binding. The ligand creates a local energetic perturbation that propagates in the form of dominolike changes in interresidue interaction pattern. There are significant changes in the nature of specific interactions (nonpolar/ polar) between interresidue contacts and accompanied side-chain reorientations that drive the major redistribution of energy. Interestingly, this internal redistribution and rewiring of side-chain interactions led to large cancellations resulting in small change in the overall enthalpy of the protein, thus making it difficult to detect experimentally. In contrast to the prevailing focus on the entropic or dynamic effects, we show that the internal redistribution and population shift in specific electrostatic interactions drive the allosteric modulation in the PDZ3 domain protein.A llosteric regulation of proteins plays a key role in physiological cell functions, biochemical and signal transduction pathways, and drug discovery (1-3). It has remained a challenge to understand how the thermodynamic perturbation caused by ligand binding at one site would propagate and modulate the structure and dynamics of distal regions of proteins. The prevailing models of structure-based allostery (4, 5) do not apply to the more recent examples of allostery without conformational change such as PDZ domain (6), CAP dimer (7), and met repressor (8). These examples have triggered the concept of dynamic allostery, where the side-chain dynamics is modulated on ligand binding and the origin has often been attributed to changes in the conformational entropy (9, 10). The modern view of allostery invokes a thermodynamic picture, where a population shift among preexisting conformational states occurs on binding the allosteric effector (11-13). It has also been suggested in the context of allostery without conformational change that "not observed does not imply that it is not there" (10) because crystallographic techniques may not resolve the relatively minor population shifts. An interesting idea has emerged that all proteins might be allosteric in nature (14).PDZ domain has been a classic model system to study single domain allostery without major structural changes (9, 15, 16) (Fig. 1A). PDZ domains are evolutionary conserved proteinprotein interaction module...

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Section: Resultsmentioning

confidence: 99%

Hidden electrostatic basis of dynamic allostery in a PDZ domain

Kumawat

Chakrabarty

2017

Proc. Natl. Acad. Sci. U.S.A.

138

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“…Such structures could potentially harbor open binding sites that are invisible in crystal structures. To this end, we isolated an ensemble of 75 structures based on rmsd clustering of GTP-bound K-RasQ61H conformers derived from previously reported MD simulations (24). Taking advantage of the small number of related compounds that we were interested in, we deliberately used a small rmsd cutoff of 1.3 Å to generate a large number of clusters and used all of the cluster centroids to include infrequently visited K-Ras conformers.…”

Section: Methodsmentioning

confidence: 99%

“…After docking and site identification, we tested whether binding at the predicted sites is viable by performing MD on selected protein-ligand complexes. The simulation details are similar to those described previously (24) and in SI Materials and Methods. By assigning different initial velocities, we generated two sets of five separate trajectories with SRJ23 bound to K-Ras at either of two preferred sites (p1 and p2).…”

Section: Methodsmentioning

confidence: 99%

“…Along this line, a recent study found a compound that binds to an open switch 1 conformation of state 1 Ras (23). In this work, we use ensembles of K-Ras obtained from molecular dynamics (MD) simulations that sample state 1 and other intermediate structures (24) to address two major questions: first, to evaluate whether the reported anticancer activity (25) of andrographolide (AGP), a diterpene from the medicinal plant Andrographis paniculata (26), and its benzylidene derivatives involves direct inhibition of Ras; and second, to use these compounds to test a unique hypothesis that prolonged inhibition of nucleotide exchange can abrogate the function of oncogenic mutant Ras. Combining data from ensemble docking, simulations, and experiments in intact cells, we show that AGP and its derivatives inhibit Ras function by preventing GEF-induced nucleotide exchange.…”

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confidence: 99%

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Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

Hocker

Cho

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

134

133

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Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several smallmolecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)-a bicyclic diterpenoid lactone isolated from Andrographis paniculata-and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP-GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP-GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.cancer | molecular dynamics | allosteric site | drug design

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“…Free energy calculations and MD simulations successfully predict the hydration state of pockets of different chemical natures (polar vs nonpolar) 22,50,51,53 , help to understand the relation between internal hydration and function as in the case of charge separation processes [54][55][56][57] , ligand binding [58][59][60] , allostery 61,62 and can be used to estimate the contribution to stability 50,63 .…”

Section: Introductionmentioning

confidence: 99%

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

et al. 2014

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In this work we address the question whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologues G-domains of different stability: the mesophilic G domain of the Elongation-Factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time-scale we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favourable for both systems, with the hyperthermophilic protein offering a slightly more favourable environment to host water molecules. We estimate that under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that at extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G-domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

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The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

Cited by 50 publications

References 79 publications

Hidden electrostatic basis of dynamic allostery in a PDZ domain

Hidden electrostatic basis of dynamic allostery in a PDZ domain

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

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