Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

Abstract

Two crystal structures of human ornithine transcarbamylase (OTCase) complexed with the substrate carbamoyl phosphate (CP) have been solved. One structure, whose crystals were prepared by substituting N-phosphonacetyl--ornithine (PALO) liganded crystals with CP, has been refined at 2.4 A / (1 A / l 0.1 nm) resolution to a crystallographic R factor of 18.4 %. The second structure, whose crystals were prepared by co-crystallization with CP, has been refined at 2.6 A / resolution to a crystallographic R factor of 20.2 %. These structures provide important new insights into substrate recognition and ligandinduced conformational changes. Comparison of these structures with the structures of OTCase complexed with the bisubstrate

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“…The degree of domain closure for the different ligand bound structures, from most open to most closed, is as follows: sulfate, AORN, CP, CP + ANOR, ACIT + SO 4 . The magnitude of domain closure is much smaller in AOTCase (1.1–2.2°) than in OTCase (4–12°)2 or ATCase (8°) 22. In both OTCase and ATCase, domain closure is accompanied by movement of the 240's loop, which brings groups into the active site to interact with the second substrate 22, 23.…”

“…In AOTCase, CP binding orders the 80's loop and brings it into a position that allows the indole ring N atom of residue Trp77 from an adjacent monomer to bind the phosphate group of CP. In OTCase, the corresponding residue provided by the adjacent monomer is either His or Gln,2, 24–27 whereas in ATCase, the adjacent monomer contributes Ser and Lys 28–30…”

“…Substrate binding also triggers a small domain closure around the active site. AOTCase can bind CP and AORN independently, in contrast to ATCase and OTCase where substrate binding is ordered 2, 30, 36, 37. However, in order for transcarbamoylation to occur, CP and AORN probably must be bound at the same time.…”

“…Previously, an argF‐like gene (herein designated as argF'), which is essential for growth in arginine‐deprived media, was identified in Bacteroides fragilis . However, its encoded protein sequence does not contain the conserved DxxSMG ornithine binding motif characteristic of OTCases 2. Furthermore, the purified protein had no detectable OTCase activity.…”

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“…The structure of E. coli UTCase is also consistent with a catabolic role. In both ATCase and anabolic OTCase, substrate binding is ordered, with CP binding first,55, 56 and binding of the second substrate causing the 240s loop to swing in and form part of the active site. The disorder of the STRTR CP binding motif in the current UTCase structure is unique, since all other transcarbamylase structures have an ordered STRTR CP binding site even in the absence of any substrates 41, 57.…”