Stabilizing a Weak Binding State for Effectors in the Human Ras Protein by Cyclen Complexes

Rosnizeck, Ina C.; Graf, Thorsten; Spoerner, Michael; Tränkle, Jens; Filchtinski, Daniel; Herrmann, Christian; Gremer, Lothar; Vetter, Ingrid R.; Wittinghofer, Alfred; König, Burkhard; Kalbitzer, Hans Robert

doi:10.1002/anie.200907002

Cited by 103 publications

(129 citation statements)

References 18 publications

Supporting

Mentioning

118

Contrasting

Order By: Relevance

“…For GTP in water we observe −42°, and in Ras, −16°, or an almost-eclipsed conformation. This agrees with the X-ray structural models of GTP bound to Ras (24,26,27), and reveals that this energetically unfavorable GTP conformation observed at cryogenic temperatures is not an artifact caused by freezing at low temperature but still prevails under physiological conditions. The eclipsed conformation of the negatively charged oxygen atoms is energetically less favorable because it causes repulsion between the γ-and β-phosphates.…”

Section: Resultssupporting

confidence: 88%

“…Such a hydrogen bond between the Tyr32 and the neighboring Ras protein is also seen in the more recent GppNHp X-ray structures: 5P21 (24) and 1CTQ (26). However, in the latest structure, 3L8Z (27), the binding niche is closed by Tyr32. In 1QRA (26), 5P21 (24), and 1CTQ (26), the crystallization symmetry is the same, with P 32 2 1 as the symmetry operator for the space group.…”

Section: Resultsmentioning

confidence: 62%

“…26) with bound GTP is available but was taken at a cryogenic temperature; GppNHp X-ray structures 5P21 (24), 1CTQ (26), and 3L8Z (27) are also available. In all structural models, the Mg 2þ in Ras is bidentately coordinated by the β-and γ-phosphates and the nonbridging oxygen atoms of the β-and γ-phosphates are in an eclipsed position.…”

Section: Resultsmentioning

confidence: 99%

“…In 1QRA (26), 5P21 (24), and 1CTQ (26), the crystallization symmetry is the same, with P 32 2 1 as the symmetry operator for the space group. In 3L8Z (27), a different crystallization symmetry was observed with H 3 2 as the symmetry operator for the space group. Our MM simulation studies show that the closed structure is energetically preferred and the calculated vibrational spectrum fits better to the experimental one (38).…”

Section: Resultsmentioning

confidence: 99%

“…This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and experimentally by X-ray crystallography (23)(24)(25)(26)(27), electron spin resonance (28)(29)(30), and FTIR spectroscopy (5,6,(31)(32)(33).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Rudack

Xia

Schlitter

et al. 2012

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

102

View full text Add to dashboard Cite

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P i by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10 5 compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10 5 compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with timeresolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ-relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α-relative to the β-and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.enzyme catalysis | reaction mechanism | free energy of activation M any cellular processes are regulated by members of the Ras superfamily. They are switched "on" by GDP-to-GTP exchange and "off" by a GTPase reaction. GTP hydrolysis is vitally important for the regulation of several signal-transduction processes in living cells (1, 2). In the case of Ras, external growth signals are transduced to the nucleus. Site-specific oncogenic mutations inhibit the GTPase reaction and the growth signal can no longer be down-regulated. This contributes to uncontrolled cell growth, eventually leading to cancer (3). Common to all members of the Ras superfamily is the catalysis of GTP hydrolysis by the G domain (4). Ras-catalyzed GTP hydrolysis is five orders of magnitude faster than in water (30 min vs. 200 d) (5). However, to control growth signals in the living cell, a further increase of five orders of magnitude in the reaction rate (30 min to 50 ms) is enabled. This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7-22) and experimentally by X-ray crystallography (23-27), electron spin resonance (28-30), and FTIR spectroscopy (5,6,(31)(32)(33).Determination of the three-dimensional structures of Ras and GAP by X-ray crystallography represents an important milestone in the understanding of t...

show abstract

Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Rudack

Xia

Schlitter

et al. 2012

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

102

View full text Add to dashboard Cite

show abstract

Structure-based Drug Design Using NMR

Jeeves

Quill

Overduin

2015

eMagRes

View full text Add to dashboard Cite

Metall‐Bis(2‐picolyl)amin‐Komplexe als Zustand‐1(T)‐Inhibitoren für aktiviertes Ras‐Protein

et al. 2012

Self Cite

View full text Add to dashboard Cite

Allosterische Stabilisierung: Metall(II)‐Cyclene sind Inhibitoren für die Ras‐ Effektor‐Wechselwirkung, da sie einen schwach Effektor‐bindenden Konformationszustand (Zustand 1(T) in Ras) stabilisieren, und binden direkt im aktiven Zentrum. Der neue Zustand‐1(T)‐Inhibitor Zn2+‐BPA (BPA=Bis(2‐picolyl)amin) bindet außerhalb der Nukleotid‐Bindetasche, stabilisiert aber trotzdem den Zustand 1(T) und kann so die Ras‐Raf‐Wechselwirkung unterbinden.

show abstract

Stabilizing a Weak Binding State for Effectors in the Human Ras Protein by Cyclen Complexes

Cited by 103 publications

References 18 publications

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Structure-based Drug Design Using NMR

Metall‐Bis(2‐picolyl)amin‐Komplexe als Zustand‐1(T)‐Inhibitoren für aktiviertes Ras‐Protein

Contact Info

Product

Resources

About