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Crystal Structure of Histidine Triad Nucleotide- Binding Protein from the Pathogenic Fungus Candida Albicans

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Crystal Structure of Histidine Triad Nucleotide- Binding Protein from the Pathogenic Fungus Candida Albicans Molecules and Cells Minireview Crystal Structure of Histidine Triad Nucleotide- Binding Protein from the Pathogenic Fungus Candida albicans Ahjin Jung1,6, Ji-Sook Yun1,6, Shinae Kim1, Sang Ryong Kim2, Minsang Shin3, Dong Hyung Cho2, Kwang Shik Choi2,4,5,*, and Jeong Ho Chang1,5,* 1Department of Biology Education, 2School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, 3Department of Mi- crobiology, School of Medicine, 4Research Institute for Dokdo and Ulleungdo Island, Kyungpook National University, 5Research Institute for Phylogenomics and Evolution, Kyungpook National University, Daegu 41566, Korea, 6These authors contributed equally to this work. *Correspondence: [email protected] (KSC); [email protected] (JHC) http://dx.doi.org/10.14348/molcells.2018.0377 www.molcells.org Histidine triad nucleotide-binding protein (HINT) is a member of evolution. Further studies are required to address the func- of the histidine triad (HIT) superfamily, which has hydrolase tional significance of variations in the C-terminal region. This activity owing to a histidine triad motif. The HIT superfamily structural analysis of CaHINT provided important insights into can be divided to five classes with functions in galactose me- the molecular aspects of evolution within the HIT superfamily. tabolism, DNA repair, and tumor suppression. HINTs are high- ly conserved from archaea to humans and function as tumor Keywords: Candida albicans, crystal structure, histidine triad, suppressors, translation regulators, and neuropathy inhibitors. HIT family, HINT Although the structures of HINT proteins from various species have been reported, limited structural information is available for fungal species. Here, to elucidate the structural features INTRODUCTION and functional diversity of HINTs, we determined the crystal structure of HINT from the pathogenic fungus Candida albi- Histidine triad nucleotide-binding protein (HINT), which con- cans (CaHINT) in complex with zinc ions at a resolution of 2.5 tains three highly conserved histidines as catalytic residues, is Å. Based on structural comparisons, the monomer of CaHINT a member of the histidine triad (HIT) protein superfamily overlaid best with HINT protein from the protozoal species (Brenner et al., 1997). Members of the HIT superfamily, Leishmania major. Additionally, structural comparisons with which contain a conserved sequence motif (H-X-H-X-H-X-X, human HINT revealed an additional helix at the C-terminus of where X represents a hydrophobic residue) in the active site, CaHINT. Interestingly, the extended C-terminal helix interact- exhibit catalytic activities, including nucleotide hydrolase, ed with the N-terminal loop (α1–β1) and with the α3 helix, phosphoramidate hydrolase, and nucleotidyltransferase ac- which appeared to stabilize the dimerization of CaHINT. In tivities (Krakowiak et al., 2004). Based on the catalytic speci- the C-terminal region, structural and sequence comparisons ficities, sequence compositions, and structural similarities of showed strong relationships among 19 diverse species from its members, this superfamily has been historically grouped archea to humans, suggesting early separation in the course into five classes: HINT, galactose-1-phosphate uridylyltrans- Received 14 September 2018; revised 5 November 2018; accepted 14 November 2018; published online 2 January, 2019 eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 56 Mol. Cells 2019; 42(1): 56-66 Crystal Structure of HINT from Candida albicans Ahjin Jung et al. ferase (GalT), fragile HIT protein (Fhit), decapping scavenger temic Candida infections (Akter et al., 2018; Schulze and (DcpS), and aprataxin (Martin et al., 2011). Sonnenborn, 2009). Although mucosal C. albicans infec- Among these five classes, GalT is known to participate in tions are common and nonlethal (Li et al., 2002; Torosan- galactose metabolism, where it catalyzes the transfer of UDP tucci et al., 2002), systemic candidiasis is associated with a from glucose-UDP to galactose (McCorvie and Timson, high mortality rate (Pfaller and Diekema, 2007) and can 2011). Lack of GalT causes hereditary galactosemia, an in- cause neutropenia and gastrointestinal mucosal damage. born error of carbohydrate metabolism (Demirbas et al., Multiple structures of HINT proteins have been deposited 2018). Fhit, which is designated as a fragile HIT protein be- in the Protein Data Bank (PDB). However, few structural cause of the high mutation rate of its gene locus in multiple studies of HINT proteins from fungal/yeast species have been cancers, has been characterized as a tumor suppressor reported. In order to expand the molecular evolutionary pool, (Huebner and Croce, 2001). Fhit has phosphoramidase ac- we determined the crystal structure of HINT from the patho- tivity and therefore participates in the hydrolysis of dinucleo- genic fungal species C. albicans. Our findings are expected tide polyphosphates (Huang et al., 2004; Varnum et al., to provide insights into the structural basis of phylogenetic 2001). DcpS is known to involved in mRNA decay by specifi- relatedness of HINT proteins from to other closely related cally cleaving methylated mRNA transcripts (Milac et al., species. 2014). A distinguishing feature of this protein is its require- ment for an additional N-terminal domain, which undergoes MATERIALS AND METHODS a large conformational change to switch between open and closed states during catalysis (Gu and Lima, 2005; Gu et al., Construction of recombinant expression plasmids 2004; Liu et al., 2002; 2004). Aprataxin participates in DNA The genes encoding CaHINT and HINT proteins from three repair systems by hydrolyzing both dinucleotide polyphos- other fungal species (Debaryomyces hansenii, Kluyveromyces phates and phosphoramidates (Martin et al., 2011). lactis, and Schizosaccharomyces pombe), were amplified by HINT belongs to the most ancient class within the HIT su- polymerase chain reaction (PCR). The amplified fragments perfamily and is well conserved from archaea to eukaryotes. were digested with the restriction enzymes (Enzynomics, In humans, three isotypes of HINT (termed the hHint family), Korea) NdeI (cat. no. R0065) and XhoI (cat. no. R0075), for i.e., Hint1, Hint2, and Hint3, have been identified (Maize et 3 h at 37℃ in a water bath. The fragments were ligated into al., 2013). Hint1 functions as a tumor suppressor by induc- pET28a and pET26b vectors using T4 ligase (cat. no. ing apoptosis independent of its enzymatic activity (Weiske M0202S; Roche, Germany) overnight in a 20℃ incubator. and Huber, 2006). Thus, Hint1 also hydrolyzes lysyl- These vectors harbored a hexahistidine (His6)-tag at either adenylate, which is produced by lysyl-tRNA synthetase, and the N- or C-terminus of the target protein. The ligated plas- mutation in Hint1 causes heritable axonal neuropathy with mids were transformed into Escherichia coli (E. coli ), strain neuromyotonia (Butland et al., 2005; Chou and Wagner, DH5α, and transformants were confirmed using colony PCR. 2007; Zimon et al., 2012). Both hHint2 and hHint3 are up- The recombinant plasmids were verified by DNA sequencing. regulated in breast, pancreatic, and colon cancers, similar to hHint1 (Martin et al., 2011). However, the biological roles of Expression and purification of recombinant proteins HINT proteins from other species have not yet been fully The resulting plasmids encoding HINT proteins were trans- characterized. formed into E. coli strain Rosetta. Cell cultures were grown The second histidine of the histidine triad in the active site at 37℃ to an OD600 of 0.6 in LB medium (cat. no. L4488; is strictly conserved and participates in catalysis with the third MBcell, Republic of Korea) containing 50 mg/L kanamycin histidine, whereas the first histidine in this motif is not in- (cat. no. A1493; Applichem, USA) and 50 mg/L chloram- volved in the reaction (Maize et al., 2013). These conserved phenicol (cat. no. A1806; Applichem). Following induction residues participate in catalysis by forming a covalent nucleo- with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; tidyl-phosphohistidyl intermediate. HINT proteins also have a cat. no. 420322; Calbiochem, Germany) and addition of conserved zinc-binding motif ‘C-X-X-C’ (where C is a cysteine 100 mM zinc chloride (cat. no. 8B4079; Junsei Chemical, residue and X is a hydrophobic residue), and a zinc ion is Tokyo), the cells were incubated for an additional 16 h at coordinated by these cysteine residues, together with the 20℃. The cultured cells were harvested by centrifugation at first histidine residue (Klein et al., 1998; Lima et al., 1996). 4500 × g for 20 min at 4℃. The cell pellet was resuspended The opportunistic pathogenic fungus Candida albicans in a buffer containing 250 mM sodium chloride (NaCl; cat. causes candidiasis (Pfaller and Diekema, 2007). Candidiasis no. A2942; Applichem), 5% glycerol (cat. no. 56515; Affy- can be classified into two categories depending on the se- metrix, USA), 0.2% Triton X-100 (cat. no. 9002931; Sigma- verity of the disease (Spampinato and Leonardi, 2013). The Aldrich, USA), 10 mM β-mercaptoethanol (cat. no. 60242; first category of candidiasis causes mucosal infections, in- BioBasic, Canada), 0.2 mM phenylmethylsulfonyl fluoride cluding infections
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