Identification ofLeishmania donovaniperoxin 14 residues required for binding the peroxin 5 receptor proteins

Abstract

Trafficking of peroxisomal targeting signal 1 (PTS1) proteins to the Leishmania glycosome is dependent on the docking of the LdPEX5 receptor to LdPEX14 on the glycosomal membrane. A combination of deletion and random mutagenesis was used to identify residues in the LdPEX14 N-terminal region that are critical for mediating the LdPEX5-LdPEX14 interaction. These studies highlighted residues 35-75 on ldpex14 as the core domain required for binding LdPEX5. Single point mutation within this core domain generally did not affect the ldpex5-(203-391)-ldpex14-(1-120) interaction; notable exceptions were substitutions at Phe40, Val46 or Phe57 which completely abolished or increased the apparent Kd value for ldpex5-(203-391) binding 30-fold. Biochemical studies revealed that these point mutations did not alter either the secondary or quaternary structure of LdPEX14 and indicated that the latter residues were critical for stabilizing the LdPEX5-LdPEX14 interaction.

The Leishmania donovani peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on peroxin 5

“…Biochemical studies with the N-terminal domain of LdPEX14 confirmed that an analogous region encompassing residues 35-75 was also sufficient for binding LdPEX5 [25]. Interestingly, the Leishmania PEX14 fragment lacked a threonine residue at position 31 (T31) which in the human PEX14 was shown to be critical for stabilizing PEX5 binding, but instead contained a serine residue (S36) at the corresponding position.…”

“…pET30b-ldpex14 (21-75) construct was generated by cloning the PCR fragment encompassing the LdPEX14 residues 21-75 into NdeI and XhoI restriction sites of pET30b vector. Generation of pUT18C-LdPEX14 and pKT25-LdPEX5 for bacterial two-hybrid experiments and pET30b-ldpex14 (1-120), pTYB12-LdPEX5, and pTYB12-ldpex5 (203-390) for protein expression were previously described [25]. The integrity of all vectors was validated by automated DNA sequencing.…”

“…Another structural difference detected between the human and Leishmania PEX14 binding domain was the ability of LdPEX14 to harboring mutations at lysine 61 (K61) (equivalent of K56 in the human PEX14) to effectively form a complex with LdPEX5. In the human PEX14, K56 plays a key role in forming the pocket that accommodates the tryptophan in the PEX5 WXXXF/Y motif and mutations of this residue are sufficient to impair PEX5 binding [21,25]. Crystal structures of the rat PEX14 N-terminal domain showed that this protein adopted a helix-turn-helix fold that crystallized in symmetric homo-dimer [26,27].…”

“…Crystal structures of the rat PEX14 N-terminal domain showed that this protein adopted a helix-turn-helix fold that crystallized in symmetric homo-dimer [26,27]. The N-terminus of LdPEX14 (ldpex14 (1-120)) has also been reported to form a stable dimer in solution [25].…”

“…Interestingly, the Leishmania PEX14 fragment lacked a threonine residue at position 31 (T31) which in the human PEX14 was shown to be critical for stabilizing PEX5 binding, but instead contained a serine residue (S36) at the corresponding position. Substitution of at S36 or deletion mutations that removed the first 39 amino acid surprisingly did not significantly impact the capacity of LdPEX14 to bind LdPEX5 [25]. Another structural difference detected between the human and Leishmania PEX14 binding domain was the ability of LdPEX14 to harboring mutations at lysine 61 (K61) (equivalent of K56 in the human PEX14) to effectively form a complex with LdPEX5.…”