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A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu.

Mercier E, Girodat D, Wieden HJ - Sci Rep (2015)

Bottom Line: The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins.Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

View Article: PubMed Central - PubMed

Affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.

ABSTRACT
The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu · GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

No MeSH data available.


Related in: MedlinePlus

Interactions between the P-loop and helix C are broken by guanine nucleotide exchange factors for EF-Tu and Ran.(a) Interactions between the P-loop and Helix C in EF-Tuwt·GTP after 10 ns of MD simulation are compared to EF-TuM112L·GTP and the crystal structure of the EF-Tu·EF-Ts complex. (b) A similar interaction between the P-loop and adjacent helix of Ran compared in X-ray structures of Ran·GDPNP (PDBID: 1IBR) and Ran·RCC1(PDBID: 1I2M). The P-loops and helices are shown in cartoon representation; sidechain and/or backbone atoms of amino acids participating in hydrogen bonds are shown as sticks; GTP and Mg2+ are represented as sticks and space-filled representation, respectively. All hydrogen atoms are omitted for clarity, and green dashed lines connect heavy atoms involved in each hydrogen bond. All superimpositions were performed using P-loop backbone atoms.
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f7: Interactions between the P-loop and helix C are broken by guanine nucleotide exchange factors for EF-Tu and Ran.(a) Interactions between the P-loop and Helix C in EF-Tuwt·GTP after 10 ns of MD simulation are compared to EF-TuM112L·GTP and the crystal structure of the EF-Tu·EF-Ts complex. (b) A similar interaction between the P-loop and adjacent helix of Ran compared in X-ray structures of Ran·GDPNP (PDBID: 1IBR) and Ran·RCC1(PDBID: 1I2M). The P-loops and helices are shown in cartoon representation; sidechain and/or backbone atoms of amino acids participating in hydrogen bonds are shown as sticks; GTP and Mg2+ are represented as sticks and space-filled representation, respectively. All hydrogen atoms are omitted for clarity, and green dashed lines connect heavy atoms involved in each hydrogen bond. All superimpositions were performed using P-loop backbone atoms.

Mentions: Conformational changes in the P-loop of EF-Tu, induced by EF-Ts binding, have been proposed to stimulate EF-Tu·nucleotide dissociation11. Increased P-loop mobility would likely accelerate these conformational changes and, therefore, promote nucleotide dissociation. The observed peptide-flip between Valine 20 and Aspartate 21 in the P-loop of EF-Tu when bound to its exchange factor EF-Ts11 complements the alternative P-loop conformations observed in our MD simulations (Fig. S2). The function of the P-loop anchor in modulating P-loop structural dynamics is supported by the observation that the P-loop anchor in EF-Tu is disrupted (Fig. 7a) in the crystal structure of the EF-Tu·EF-Ts complex11. This suggests that EF-Ts may have evolved to act on this conserved element to increase P-loop mobility during nucleotide dissociation. Similarly, X-ray crystal structures of Ran in complex with GDPNP or its GEF RCC1 reveal that GEF binding is concomitant with breaking the P-loop anchor (Fig. 7b) in this G-protein as well28. Interestingly, crystal structures of Ras and Rac1 bound to their respective GEFs demonstrate that disruption of the P-loop anchor interactions is not a requirement for GEF binding729, but it seems likely that some GEFs have evolved in the context of this P-loop anchor in order to modulate P-loop structural dynamics and promote nucleotide dissociation.


A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu.

Mercier E, Girodat D, Wieden HJ - Sci Rep (2015)

Interactions between the P-loop and helix C are broken by guanine nucleotide exchange factors for EF-Tu and Ran.(a) Interactions between the P-loop and Helix C in EF-Tuwt·GTP after 10 ns of MD simulation are compared to EF-TuM112L·GTP and the crystal structure of the EF-Tu·EF-Ts complex. (b) A similar interaction between the P-loop and adjacent helix of Ran compared in X-ray structures of Ran·GDPNP (PDBID: 1IBR) and Ran·RCC1(PDBID: 1I2M). The P-loops and helices are shown in cartoon representation; sidechain and/or backbone atoms of amino acids participating in hydrogen bonds are shown as sticks; GTP and Mg2+ are represented as sticks and space-filled representation, respectively. All hydrogen atoms are omitted for clarity, and green dashed lines connect heavy atoms involved in each hydrogen bond. All superimpositions were performed using P-loop backbone atoms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286738&req=5

f7: Interactions between the P-loop and helix C are broken by guanine nucleotide exchange factors for EF-Tu and Ran.(a) Interactions between the P-loop and Helix C in EF-Tuwt·GTP after 10 ns of MD simulation are compared to EF-TuM112L·GTP and the crystal structure of the EF-Tu·EF-Ts complex. (b) A similar interaction between the P-loop and adjacent helix of Ran compared in X-ray structures of Ran·GDPNP (PDBID: 1IBR) and Ran·RCC1(PDBID: 1I2M). The P-loops and helices are shown in cartoon representation; sidechain and/or backbone atoms of amino acids participating in hydrogen bonds are shown as sticks; GTP and Mg2+ are represented as sticks and space-filled representation, respectively. All hydrogen atoms are omitted for clarity, and green dashed lines connect heavy atoms involved in each hydrogen bond. All superimpositions were performed using P-loop backbone atoms.
Mentions: Conformational changes in the P-loop of EF-Tu, induced by EF-Ts binding, have been proposed to stimulate EF-Tu·nucleotide dissociation11. Increased P-loop mobility would likely accelerate these conformational changes and, therefore, promote nucleotide dissociation. The observed peptide-flip between Valine 20 and Aspartate 21 in the P-loop of EF-Tu when bound to its exchange factor EF-Ts11 complements the alternative P-loop conformations observed in our MD simulations (Fig. S2). The function of the P-loop anchor in modulating P-loop structural dynamics is supported by the observation that the P-loop anchor in EF-Tu is disrupted (Fig. 7a) in the crystal structure of the EF-Tu·EF-Ts complex11. This suggests that EF-Ts may have evolved to act on this conserved element to increase P-loop mobility during nucleotide dissociation. Similarly, X-ray crystal structures of Ran in complex with GDPNP or its GEF RCC1 reveal that GEF binding is concomitant with breaking the P-loop anchor (Fig. 7b) in this G-protein as well28. Interestingly, crystal structures of Ras and Rac1 bound to their respective GEFs demonstrate that disruption of the P-loop anchor interactions is not a requirement for GEF binding729, but it seems likely that some GEFs have evolved in the context of this P-loop anchor in order to modulate P-loop structural dynamics and promote nucleotide dissociation.

Bottom Line: The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins.Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

View Article: PubMed Central - PubMed

Affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.

ABSTRACT
The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu · GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

No MeSH data available.


Related in: MedlinePlus