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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

His22 and Met112 form close contacts in the secondary shell surrounding the P-loop of EF-Tu.The model of E. coli EF-Tu·GTP is shown in cartoon representation after 10 ns of molecular dynamics simulation. Bound GTP and Mg2+ are shown as sticks and space-filling, respectively, both coloured silver. The backbone atoms of the P-loop are shown as sticks and hydrogen bonds between P-loop amide hydrogen atoms and phosphate oxygen atoms of GTP are shown as green dashed lines. His22 (black) of the P-loop and Met112 (red) of the adjacent coil are shown in space-filling representation.
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f1: His22 and Met112 form close contacts in the secondary shell surrounding the P-loop of EF-Tu.The model of E. coli EF-Tu·GTP is shown in cartoon representation after 10 ns of molecular dynamics simulation. Bound GTP and Mg2+ are shown as sticks and space-filling, respectively, both coloured silver. The backbone atoms of the P-loop are shown as sticks and hydrogen bonds between P-loop amide hydrogen atoms and phosphate oxygen atoms of GTP are shown as green dashed lines. His22 (black) of the P-loop and Met112 (red) of the adjacent coil are shown in space-filling representation.

Mentions: Based on the available EF-Tu structures, sequence conservation, and our own MD simulations (vide infra), we have identified two amino acid positions that are likely to be critical for the structural dynamics and overall flexibly of the P-loop in EF-Tu. Histidine 22 is located in the P-loop (Fig. 1), is 100% conserved among bacterial EF-Tus21, and has previously been shown to form a transient hydrogen bond to the adjacent second shell residue Aspartate 10922. Although important for EF-Ts-modulated nucleotide exchange, this hydrogen bond is not important for fine-tuning nucleotide binding in the EF-Tu·GTP complex22. Interestingly, Histidine 22 is in close proximity to a highly (>99%) conserved Methionine in position 112 (Fig. 1). We therefore reasoned that by packing against Methionine in position 112 Histidine 22 might play a role in limiting the internal structural dynamics (flexibility) of the P-loop as well as restricting the mobility of the P-loop as a whole. To address this we have constructed a Histidine 22 substitution variant containing Glycine in this position (EF-TuH22G) to increase the internal flexibility and mobility of the P-loop. Also, we have introduced Glycine or Alanine in position 112 (EF-TuM112G and EF-TuM112A respectively) to increase the mobility of Histidine 22 and, in turn, the P-loop. We also constructed a Leucine variant (EF-TuM112L) to control for altering the space requirements of the side chain in position 112 without affecting the contacts between this position and the P-loop.


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)

His22 and Met112 form close contacts in the secondary shell surrounding the P-loop of EF-Tu.The model of E. coli EF-Tu·GTP is shown in cartoon representation after 10 ns of molecular dynamics simulation. Bound GTP and Mg2+ are shown as sticks and space-filling, respectively, both coloured silver. The backbone atoms of the P-loop are shown as sticks and hydrogen bonds between P-loop amide hydrogen atoms and phosphate oxygen atoms of GTP are shown as green dashed lines. His22 (black) of the P-loop and Met112 (red) of the adjacent coil are shown in space-filling representation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: His22 and Met112 form close contacts in the secondary shell surrounding the P-loop of EF-Tu.The model of E. coli EF-Tu·GTP is shown in cartoon representation after 10 ns of molecular dynamics simulation. Bound GTP and Mg2+ are shown as sticks and space-filling, respectively, both coloured silver. The backbone atoms of the P-loop are shown as sticks and hydrogen bonds between P-loop amide hydrogen atoms and phosphate oxygen atoms of GTP are shown as green dashed lines. His22 (black) of the P-loop and Met112 (red) of the adjacent coil are shown in space-filling representation.
Mentions: Based on the available EF-Tu structures, sequence conservation, and our own MD simulations (vide infra), we have identified two amino acid positions that are likely to be critical for the structural dynamics and overall flexibly of the P-loop in EF-Tu. Histidine 22 is located in the P-loop (Fig. 1), is 100% conserved among bacterial EF-Tus21, and has previously been shown to form a transient hydrogen bond to the adjacent second shell residue Aspartate 10922. Although important for EF-Ts-modulated nucleotide exchange, this hydrogen bond is not important for fine-tuning nucleotide binding in the EF-Tu·GTP complex22. Interestingly, Histidine 22 is in close proximity to a highly (>99%) conserved Methionine in position 112 (Fig. 1). We therefore reasoned that by packing against Methionine in position 112 Histidine 22 might play a role in limiting the internal structural dynamics (flexibility) of the P-loop as well as restricting the mobility of the P-loop as a whole. To address this we have constructed a Histidine 22 substitution variant containing Glycine in this position (EF-TuH22G) to increase the internal flexibility and mobility of the P-loop. Also, we have introduced Glycine or Alanine in position 112 (EF-TuM112G and EF-TuM112A respectively) to increase the mobility of Histidine 22 and, in turn, the P-loop. We also constructed a Leucine variant (EF-TuM112L) to control for altering the space requirements of the side chain in position 112 without affecting the contacts between this position and the P-loop.

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