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A computational study of a recreated G protein-GEF reaction intermediate competent for nucleotide exchange: fate of the Mg ion.

Hamida-Rebaï MB, Robert CH - PLoS ONE (2010)

Bottom Line: We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence.The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction.The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

View Article: PubMed Central - PubMed

Affiliation: CNRS Institute of Biochemistry and Molecular and Cellular Biology (IBBMC), Université Paris-Sud 11, Orsay, France.

ABSTRACT
Small G-proteins of the superfamily Ras function as molecular switches, interacting with different cellular partners according to their activation state. G-protein activation involves the dissociation of bound GDP and its replacement by GTP, in an exchange reaction that is accelerated and regulated in the cell by guanine-nucleotide exchange factors (GEFs). Large conformational changes accompany the exchange reaction, and our understanding of the mechanism is correspondingly incomplete. However, much knowledge has been derived from structural studies of blocked or inactive mutant GEFs, which presumably closely represent intermediates in the exchange reaction and yet which are by design incompetent for carrying out the nucleotide exchange reaction. In this study we have used comparative modelling to recreate an exchange-competent form of a late, pre-GDP-ejection intermediate species in Arf1, a well-characterized small G-protein. We extensively characterized three distinct models of this intermediate using molecular dynamics simulations, allowing us to address ambiguities related to the mutant structural studies. We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence. The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction. The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

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Energy of interaction between Arf1 and Mg.Interaction energy between Arf1 and Mg in the molecular dynamics simulations for the Mg-containing model complexes IIm1 and IIm2 (top and middle sets of lines, respectively) and for the reference Arf1-GDP-Mg system (1hur) seen at bottom, presented as a function of simulation time.
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pone-0009142-g003: Energy of interaction between Arf1 and Mg.Interaction energy between Arf1 and Mg in the molecular dynamics simulations for the Mg-containing model complexes IIm1 and IIm2 (top and middle sets of lines, respectively) and for the reference Arf1-GDP-Mg system (1hur) seen at bottom, presented as a function of simulation time.

Mentions: Limited positional fluctuations do not necessarily reflect a strong interaction between Mg and Arf1. In Arf1-GDP alone, the energy of interaction measured between Arf1 and Mg (Figure 3) was strongly negative; while in the intermediate complexes the interaction became somewhat (IIm2) or substantially (IIm1) positive. Two factors contribute to this effect. First, in the inactive Arf1-GDP complex, Arf1 residues Glu54 and Asp67 interact closely (carboxylate distances of 3.7 and 4.6 A, respectively) with the Mg. In the transformation to intermediate II, the so-called “interswitch” beta-hairpin containing both residues is displaced [6], as reflected in Figure 2, which modifies the respective distances to at least 16.0 and 3.8 A and thus removing a source of Arf1's electrostatic stabilization of the Mg. Second, in the inactive complex Arf1-GDP, the Mg interacts with Arf residue Thr31 and with one of the GDP beta phosphate oxygens [26]. The Thr31OG-Mg distance remains on the order of 2.3 A in the inactive Arf1-GDP complex and in intermediate I of the exchange reaction. In both models IIm1 and IIm2, this interaction is modified. In IIm2, the interaction is lost due to the initial Mg ion placement in this system. In IIm1, in which the Mg was initially placed in the position seen in the inactive Arf1-GDP complex, as well as in intermediate I, the Thr31OG distance was found to increase to 3.1 A as the Mg drew closer to the second beta-phosphate oxygen during the equilibration period. The ion thus gained electrostatic interaction with the GDP at the expense of its interaction with Arf1. The motor for this modification appears to be the approach of the GEF. In particular, the N-terminal of GEF helix 7, which could contribute via a helix dipole effect, is much closer to the Mg binding site (5 A) in intermediate II than in intermediate I (12 A) due to the maturing of the Arf1-GEF interface. Indeed, GEF residue Gln158 alone, at the N-terminal of this helix, was seen to add +5 kcal/mol to the interaction energy with the Mg.


A computational study of a recreated G protein-GEF reaction intermediate competent for nucleotide exchange: fate of the Mg ion.

Hamida-Rebaï MB, Robert CH - PLoS ONE (2010)

Energy of interaction between Arf1 and Mg.Interaction energy between Arf1 and Mg in the molecular dynamics simulations for the Mg-containing model complexes IIm1 and IIm2 (top and middle sets of lines, respectively) and for the reference Arf1-GDP-Mg system (1hur) seen at bottom, presented as a function of simulation time.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823772&req=5

pone-0009142-g003: Energy of interaction between Arf1 and Mg.Interaction energy between Arf1 and Mg in the molecular dynamics simulations for the Mg-containing model complexes IIm1 and IIm2 (top and middle sets of lines, respectively) and for the reference Arf1-GDP-Mg system (1hur) seen at bottom, presented as a function of simulation time.
Mentions: Limited positional fluctuations do not necessarily reflect a strong interaction between Mg and Arf1. In Arf1-GDP alone, the energy of interaction measured between Arf1 and Mg (Figure 3) was strongly negative; while in the intermediate complexes the interaction became somewhat (IIm2) or substantially (IIm1) positive. Two factors contribute to this effect. First, in the inactive Arf1-GDP complex, Arf1 residues Glu54 and Asp67 interact closely (carboxylate distances of 3.7 and 4.6 A, respectively) with the Mg. In the transformation to intermediate II, the so-called “interswitch” beta-hairpin containing both residues is displaced [6], as reflected in Figure 2, which modifies the respective distances to at least 16.0 and 3.8 A and thus removing a source of Arf1's electrostatic stabilization of the Mg. Second, in the inactive complex Arf1-GDP, the Mg interacts with Arf residue Thr31 and with one of the GDP beta phosphate oxygens [26]. The Thr31OG-Mg distance remains on the order of 2.3 A in the inactive Arf1-GDP complex and in intermediate I of the exchange reaction. In both models IIm1 and IIm2, this interaction is modified. In IIm2, the interaction is lost due to the initial Mg ion placement in this system. In IIm1, in which the Mg was initially placed in the position seen in the inactive Arf1-GDP complex, as well as in intermediate I, the Thr31OG distance was found to increase to 3.1 A as the Mg drew closer to the second beta-phosphate oxygen during the equilibration period. The ion thus gained electrostatic interaction with the GDP at the expense of its interaction with Arf1. The motor for this modification appears to be the approach of the GEF. In particular, the N-terminal of GEF helix 7, which could contribute via a helix dipole effect, is much closer to the Mg binding site (5 A) in intermediate II than in intermediate I (12 A) due to the maturing of the Arf1-GEF interface. Indeed, GEF residue Gln158 alone, at the N-terminal of this helix, was seen to add +5 kcal/mol to the interaction energy with the Mg.

Bottom Line: We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence.The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction.The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

View Article: PubMed Central - PubMed

Affiliation: CNRS Institute of Biochemistry and Molecular and Cellular Biology (IBBMC), Université Paris-Sud 11, Orsay, France.

ABSTRACT
Small G-proteins of the superfamily Ras function as molecular switches, interacting with different cellular partners according to their activation state. G-protein activation involves the dissociation of bound GDP and its replacement by GTP, in an exchange reaction that is accelerated and regulated in the cell by guanine-nucleotide exchange factors (GEFs). Large conformational changes accompany the exchange reaction, and our understanding of the mechanism is correspondingly incomplete. However, much knowledge has been derived from structural studies of blocked or inactive mutant GEFs, which presumably closely represent intermediates in the exchange reaction and yet which are by design incompetent for carrying out the nucleotide exchange reaction. In this study we have used comparative modelling to recreate an exchange-competent form of a late, pre-GDP-ejection intermediate species in Arf1, a well-characterized small G-protein. We extensively characterized three distinct models of this intermediate using molecular dynamics simulations, allowing us to address ambiguities related to the mutant structural studies. We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence. The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction. The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

Show MeSH
Related in: MedlinePlus