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Related Experiment Videos

Mutational analyses support a model for the HRV2 2A proteinase

W Sommergruber1, J Seipelt, F Fessl

  • 1Department Cell Biology, Boehringer-Ingelheim Research Vienna, Bender and Co., Austria.

Virology
|August 4, 1997
PubMed
Summary
This summary is machine-generated.

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Human rhinovirus 2 proteinase 2A has a three-dimensional model revealing a two-domain structure. Zinc binding is crucial for its activity, with specific residues forming the catalytic site and substrate recognition regions.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Virology

Background:

  • Human rhinovirus 2 proteinase 2A (HRV2 2A) is a cysteine proteinase essential for viral replication.
  • A tightly bound zinc ion is hypothesized to be critical for HRV2 2A's structural integrity and function.

Purpose of the Study:

  • To establish a three-dimensional model of HRV2 2A.
  • To investigate the roles of specific residues in catalytic activity, substrate binding, and zinc coordination.

Main Methods:

  • Sequence alignments with trypsin-like serine proteinases and elastase were used to build a 3D model.
  • Site-directed mutagenesis of HRV2 2A was performed, and mutants were expressed in bacteria.
  • Enzyme activity (cis and trans) of mutants was assessed.
  • Atom emission spectroscopy and alkylation procedures were used to analyze zinc content and disulphide bridges.

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Main Results:

  • A two-domain model for HRV2 2A was proposed, with distinct faces for the catalytic site and zinc-binding motif.
  • Specific residues (Gly123, Gly124, Thr121, Cys101) are implicated in the substrate binding pocket architecture.
  • Residues Tyr85 and Tyr86 are suggested to be involved in substrate recognition.
  • An extensive C-terminal helix, involving residues Asp132, Arg134, Phe130, and Phe136, is crucial for proteinase activity.
  • The zinc-binding motif (Cys52, Cys54, Cys112, His114) was identified, and mutations within it inactivated the enzyme.
  • Zinc was absent in HRV2 2A mutants where His114 was replaced by Asn.
  • The absence of disulphide bridges was confirmed.

Conclusions:

  • The established 3D model provides insights into the structural and functional organization of HRV2 2A.
  • The zinc ion, coordinated by Cys52, Cys54, Cys112, and His114, is essential for HRV2 2A activity.
  • Specific residues contribute to substrate binding, recognition, and catalytic function, highlighting the enzyme's complex architecture.