Substitution of conserved amino acids within regions of Mpr1 required for Zn2+ coordination and catalytic activity.
(A) The scaled, schematic representation of the predicted structural features of Mpr1 indicates the signal sequence (SS), the prodomain containing a conserved FTP motif and the zinc-binding sites within the M36 catalytic domain. (B) The Mpr1 protein sequence alignment across fungal species revealed strong consensus among amino acids required for coordinating zinc and for catalytic activity (HExxH and ExxxD). (H, histidine; E, glutamic acid; D, aspartic acid). (C) Amino acid sequence depiction of wild type Mpr1 and three zinc-binding site mutants used in the study. MPR1wt is the reconstituted strain (mpr1Δ::MPR1) with conserved H586E587xxH590 and E616xxxD620 regions. The MPR1QG and MPR1QGA mutants contain amino acid substitutions within the HExxH consensus region as follows: H586Q, and E587G or H586Q, E587G and H590A. The MPR1QGAAA mutant contains substitutions in both HExxH and ExxxD consensus regions as follows: H586Q, E587G, H590A, E616A, D620A. (Q, glutamine; G, glycine; A, alanine). (D) The growth curves of C. neoformans strains expressing the MPR1 mutants demonstrated no significant difference in the growth rates at 37°C suggesting that expression of the MPR1 mutants did not negatively impact the viability of C. neoformans. The description of the MPR1 mutants is detailed above. The H99 strain represents a wild type strain of C. neoformans MPR1WT. The mpr1Δ deletion strain (constructed in an H99 background) was used as the background strain for the expression of each MPR1 mutant.