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Related Experiment Videos

Nucleotide-binding sites in V-type Na+-ATPase from Enterococcus hirae.

Takeshi Murata1, Yasushi Yoshikawa, Toshiaki Hosaka

  • 1The Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK.

Journal of Biochemistry
|November 6, 2002
PubMed
Summary

Enterococcus hirae V-ATPase exhibits N-ethylmaleimide (NEM) resistance due to an alanine substitution. Replacing this alanine with cysteine restored NEM sensitivity, revealing key structural insights into V-ATPase function.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Most vacuolar-type ATPases (V-ATPases) are sensitive to N-ethylmaleimide (NEM), a property linked to a cysteine residue.
  • Enterococcus hirae V-ATPase presents an exception, displaying resistance to NEM.

Purpose of the Study:

  • To investigate the molecular basis for NEM resistance in Enterococcus hirae V-ATPase.
  • To model the three-dimensional structure of the NtpB subunit and predict nucleotide-binding sites.

Main Methods:

  • Amino acid sequence alignment of NtpA to identify substitutions.
  • Site-directed mutagenesis to substitute alanine with cysteine.
  • Homology modeling based on bovine F(1)-ATPase structure to determine NtpB 3D structure.
  • Experimental validation of nucleotide-binding sites.

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Main Results:

  • The NEM-sensitive cysteine in other V-ATPases is replaced by alanine in E. hirae V-ATPase (NtpA).
  • Substitution of this alanine with cysteine rendered the E. hirae V-ATPase NEM-sensitive.
  • The 3D structure of the NtpB subunit was successfully modeled, showing resemblance to the alpha subunit of bovine F(1)-ATPase.
  • Experimental data confirmed approximately six nucleotide-binding sites on the E. hirae V-ATPase, despite the absence of a canonical P-loop in NtpB.

Conclusions:

  • The alanine residue at the NEM-sensitive site is responsible for the NEM resistance of E. hirae V-ATPase.
  • Structural modeling provides insights into the V-ATPase subunit structure and nucleotide binding.
  • E. hirae V-ATPase possesses multiple nucleotide-binding sites crucial for its function.