Critical Roles of Interactions among Switch I-preceding Residues and between Switch II and Its Neighboring α-Helix in Conformational Dynamics of the GTP-bound Ras Family Small GTPases

Matsumoto, Kousuke; Shima, Fumi; Muraoka, Shin; Araki, Mitsugu; Hu, Lizhi; Ijiri, Yuichi; Hirai, Rina; Liao, Jingling; Yoshioka, Tatsuya; Kumasaka, Takashi; Yamamoto, Masaki; Tamura, Atsuo; Kataoka, Tohru

doi:10.1074/jbc.m110.204933

Cited by 15 publications

(16 citation statements)

References 31 publications

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“…S6C). 31 P NMR measurements indicate that mutations that disrupt hydrogen bonds along this network alter interactions between H-Ras and the GTP-analogue (36). In addition, mutations of H-Ras paralogs that abut or are located on helix 3 (Arg68Thr, Asp69Asn, Met72Ile, Asp92Asn, Asp92Tyr, Arg97Gly) have been identified in cancers (37), supporting the idea that the network is critical for the function of H-Ras (Fig.…”

Section: Discussionmentioning

confidence: 55%

Accessing protein conformational ensembles using room-temperature X-ray crystallography

Fraser

Bedem

Samelson

et al. 2011

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

556

471

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Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.protein conformational dynamics | energy landscape | Ringer | qFit M acromolecular X-ray crystallographic diffraction experiments provide powerful insights into the relationship between structure and biological function. Although macromolecules populate vast ensembles of alternative conformational substates (1), crystallographic models depicting the major average conformation have provided foundational ideas about the mechanisms of biochemical reactions. By slowing radiation damage to the sample, crystal cooling has catalyzed a revolution in structural biology, enabling structure determinations from tiny crystals using bright synchrotron X-ray sources (2-4). It is estimated that more than 95% of the >65;000 crystal structures deposited in the Protein Data Bank (PDB) are based on cryogenic data (5).Crystal cooling is generally thought to introduce little bias in the functional interpretation of structural results. Some investigators have suggested that the standard practice of plunging crystals into liquid nitrogen and collecting X-ray diffraction data at 100 K traps a representative set of conformations populated at room temperature (6, 7). In contrast, early structural comparisons by Petsko, Frauenfelder, and colleagues indicated that cooling myoglobin crystals causes a small reduction in the protein volume due to anisotropic displacements of atomic positions and subtle changes of contacts between α-helices (8). A landmark study of crystalline ribonuclease A (RNaseA) by Tilton, Petsko, and coworkers revealed diverse temperature-dependent changes in the average structure, ranging from shrinkage at low temperatures to increased loop disorder at 47°C (9). A recent analysis comparing 15 room-temperature and cryogenic crystal structures documented that cryocooling generally increas...

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Section: Discussionmentioning

confidence: 55%

Accessing protein conformational ensembles using room-temperature X-ray crystallography

Fraser

Bedem

Samelson

et al. 2011

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

556

471

View full text Add to dashboard Cite

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“…We suggest that the differences in dynamic changes observed for EF-Tu AV-165/314 and EF-Tu AV-47/314 versus EF-Tu AV-33/315 result from differential mutation and labeling effects on the dynamic equilibrium between the EF-Tu structural extremes. All three EF-Tu mutants in this study have mutations positioned spatially close either to switch I (positions 33 and 47) or to helix C (position 165) and, in the related protein, ras p21, mutations in the corresponding regions are known to affect the balance between two GTP-bound conformations ( 43 , 44 ). Similarly, a mutation in the switch II region causes the EF-Tu·GDP complex to adopt an ‘EF-Tu·GTP’-like behaviour ( 45 ).…”

Section: Discussionmentioning

confidence: 99%

Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

Kavaliauskas

Chen

Liu

et al. 2018

Nucleic Acids Research

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The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal has never been tested directly. Here, we apply single-molecule total internal reflection fluorescence microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. Our studies show that GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution ‘GTP’ to the ‘GDP’ structure. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The structural transition of EF-Tu on the ribosome is slower when aa-tRNA binds to a cognate versus a near-cognate codon. The resulting longer residence time of EF-Tu on the ribosome may be important for promoting accommodation of the cognate aminoacyl-tRNA into the A-site.

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“…It was revealed that state 1 assumes an open structure distinguishable from state 2 by the loss of the direct and Mg 2+ -coordinated indirect hydrogen-bonding interactions of Thr-35 in switch I with the c-phosphate of GppNHp, which causes marked deviation away from the guanine nucleotide and conformational instability of the switch I loop [6]. Further analyses showed the importance of the Gly-60-c-phosphate direct hydrogen-bonding interaction and the switch II-a3-helix interaction in stabilizing the state 2 conformation [6,10]. In particular, H-RasT35S-GppNHp yielded two distinct state 1 crystal structures, form 1 and form 2, showing the existence of polysterism in switch I and switch II [6].…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of the state 1 conformations of the GTP‐bound H‐Ras protein and its oncogenic G12V and Q61L mutants

et al. 2012

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a b s t r a c t GTP-bound Ras adopts two interconverting conformations, ''inactive'' state 1 and ''active'' state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the c-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.

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Critical Roles of Interactions among Switch I-preceding Residues and between Switch II and Its Neighboring α-Helix in Conformational Dynamics of the GTP-bound Ras Family Small GTPases

Abstract: GTP-bound forms of Ras family small GTPases

Cited by 15 publications

References 31 publications

Accessing protein conformational ensembles using room-temperature X-ray crystallography

Accessing protein conformational ensembles using room-temperature X-ray crystallography

Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

Crystal structures of the state 1 conformations of the GTP‐bound H‐Ras protein and its oncogenic G12V and Q61L mutants

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