Modelling and Mutational Evidence Identify the Substrate Binding Site and Functional Elements in APC Amino Acid Transporters

Mol Membr Biol. 2009 Aug;26(5):356-70. doi: 10.1080/09687680903170546. Epub 2009 Aug 7.

Abstract

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Transport Systems / chemistry
  • Amino Acid Transport Systems / genetics*
  • Amino Acid Transport Systems / metabolism*
  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Aspergillus nidulans / genetics
  • Binding Sites
  • Computer Simulation
  • DNA Mutational Analysis
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / genetics
  • Sequence Alignment
  • Sequence Homology, Amino Acid

Substances

  • Amino Acid Transport Systems
  • Amino Acids
  • Escherichia coli Proteins
  • Fungal Proteins