Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.


Introduction
The acquisition of the mitochondrial endosymbiont and its evolution into the mitochondrion were key events in the evolution of eukaryotes [1]. During this process, most of the protomitochondrial genome was either lost or transferred to the nucleus of the host cell [2]. As a consequence, most mitochondrial proteins are host-nuclear encoded and must be specifically targeted to the organelle where they function. In the best understood system, Nterminal extensions attached to mitochondrial matrix proteins are specifically recognised by receptors on the mitochondrial surface, and the preproteins are subsequently imported by translocases of the outer and inner mitochondrial membranes [3]. A final step in the import process is the removal of the N-terminal extension, by the mitochondrial processing peptidase (MPP) [4], to prevent it from interfering with protein function and/or stability [5]. The MPP comprises a catalytic bMPP subunit that binds a zinc cation using amino acid residues of the conserved motif HXXEHX 76 E [6], and a regulatory aMPP subunit with a flexible glycine-rich loop that is important for substrate recognition [7]. The two subunits together form a negatively charged cavity that accommodates and immobilizes presequences during processing [6]. The activity of MPP thus requires the cooperative action of both subunits; neither subunit is functional alone [6,8].
Mitochondrial targeting presequences are characterized by the ability to form a positively charged amphipathic alpha helix, but otherwise show little primary sequence conservation [6]. Their most prominent common feature is the presence of a cleavage motif, which determines the peptide bond to be cleaved by the processing peptidase. The cleavage motif includes a positively charged residue, typically arginine, at the -2 or -3 position from the cleavage site (P 2 or P 3 ), which is followed by hydrophobic (P 1 9) and hydrophilic (P 2 9, P 3 9) residues [9]. Mutational analyses indicate that the P 2 (P 3 ) arginine plays a key role in the recognition of the processing site by MPP and interacts with the glutamate of the bMPP active site [9]. In addition, there are one or more basic amino acid residue(s) N-terminally distal from the processing site that bind to acidic residues of the MPP cavity and stabilize the substrate-MPP complex [10].
Mitosomes and hydrogenosomes are highly reduced versions of mitochondria that are found in diverse parasitic or free-living unicellular eukaryotes inhabiting oxygen-poor or intracellular niches [1]. The organelles found in human parasites Giardia intestinalis and Trichomonas vaginalis lack a genome so all of their proteins are encoded by the nuclear genome and must be imported [1]. Some hydrogenosomal and mitosomal proteins have N-terminal extensions that are reminiscent of the presequences that direct proteins into mitochondria and they contain distinguishable cleavage motifs [11,12]. This suggests that the Giardia and Trichomonas organelles may also contain an MPP-like enzyme. A single gene coding for a putative processing peptidase has been found in the genome of G. intestinalis [13] and the gene product has been shown to localize in mitosomes [14]. The primary structure of GPP is highly divergent from mitochondrial homologues, with only 13.1% identity and 29.7% similarity to the bMPP of Saccharomyces cerevisiae. A single gene for a bMPP homologue (20.9% identity and 42.9% similarity to S. cerevisiae bMPP) was also recently identified in the genome of T. vaginalis [15]. In this case, functional data were presented suggesting that the hydrogenosomal processing peptidase (bHPP) functioned as a homodimeric enzyme [15]. No aMPP homologue was detected, although a protein rich in glycine amino acid residues (GRLP), that shares a limited similarity with the glycine-rich loop of aMPP, was located to T. vaginalis hydrogenosomes. However, GRLP was reported not to stimulate bHPP activity in vitro [15].
The progenitor of MPP was probably a monomeric aproteobacterial peptidase, similar to the recently described Rickettsia prowazekii processing peptidase (RPP) [16]. During the evolution of mitochondria, gene duplication and subunit specialization gave rise to the heterodimeric a/bMPP, which is now present in the mitochondrial matrix or integrated as the core I and II subunits of the cytochrome bc1 complex in the inner mitochondrial membrane [6]. The single subunit structure of GPP and HPP [15] could thus reflect retention of the ancestral form of organization, or reductive evolution from the classic MPP heterodimer. It has also been suggested that the Giardia protein may have had a separate origin by lateral gene transfer from a bacterium other than the mitochondrial endosymbiont [13]. Here we show that GPP functions as a monomer consisting of a single bMPP homologue while HPP, like classical MPP, is fully active only upon heterodimerization of an a and b subunit. Based upon phylogenetic and functional analyses we infer that the unique monomeric structure of the Giardia mitosomal processing peptidase GPP, is the result of reductive, substrate-driven evolution from a heterodimeric progenitor enzyme.

Phylogenetic analyses of GPP, bHPP and GRLP
To investigate the origins of the MPP-like proteins of Giardia and Trichomonas and the Trichomonas GRLP we carried out a phylogenetic analysis. As these proteins are heterogeneous for their amino acid compositions, and because a failure to accommodate such heterogeneity can lead to incorrect trees [17], we used a recently described node-discreet-compositionalheterogeneity method to analyze the data [17]. A heterogeneous model comprising 10 composition vectors was found sufficient to produce data of similar composition to the original sequences, as judged by Bayesian posterior predictive simulation [17] (Fig. S1).
Phylogenetic analyses using this model support the hypothesis that GPP, bHPP and bMPP share a common origin. This result contrasts with a previous analysis, using a poorly fitting composition homogeneous model, when GPP was reported to have no phylogenetic affinity with either MPP or the aproteobacteria [13]. The position of the GPP among bMPP, together with the presence of the catalytic motif HXXEHX 76 E, are consistent with the protein being a bMPP-like peptidase (bGPP), and not an aMPP-like protein as currently annotated [13]. Importantly, these data, together with the absence of an aMPPlike protein coding sequence on the Giardia genome, support the hypothesis that the single subunit structure of GPP results from reductive evolution including loss of an aMPP-like subunit. The alternative possibility, that the simple GPP structure reflects retention of the ancestral form of organization, is not supported by our analyses. Our results suggest that aMPP and bMPP probably arose once by a primordial gene duplication at the base of eukaryotes, and that all MPP-like proteins share common ancestry with single subunit enzymes from a-proteobacteria, consistent with an origin from the mitochondrial endosymbiont (Fig. 1A). Notably, our analyses show that the T. vaginalis GRLP is part of the aMPP clade, suggesting that, contrary to previous claims [15], T. vaginalis may possess a functional homologue (GRLP) of aMPP (henceforth aHPP).

GPP functions as a b monomer while HPP forms an a/b heterodimer
To investigate the functionality of the bGPP, bHPP and aHPPlike proteins, we expressed them in E. coli. The recombinant bGPP processed the N-terminal extensions of Giardia mitosomal ferredoxin (Gifdx) and the iron-sulphur cluster scaffold proteins (GiiscU and GiiscA). The processing activity was demonstrated as a shift in the substrate gel mobility and the cleavage sites were identified by N-terminal amino acid sequencing of the cleaved products (Fig. 2). The activity of the recombinant bGPP was inhibited by the chelator EDTA, and activity was also lost when the first glutamate of the HXXEHX 76 E motif was mutated to glutamine (Fig. 3). These data indicate that the bGPP is an active metallopeptidase

Author Summary
In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called a or b, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis. The Giardia enzyme is unique among eukaryotes because it has undergone reductive evolution to function efficiently as a single b-subunit monomer. We also show that the recent claim that the equivalent enzyme in the hydrogenosome, another type of reduced mitochondrion of the human parasite Trichomonas vaginalis, functions as a homodimer of two b-subunits, is not supported. The Trichomonas enzyme requires both an aand a b-subunit to function most efficiently. Computational analysis of the Giardia and Trichomonas enzymes reveals that their structures and surface charge distributions have coevolved to match the peculiar properties of the targeting signals that they process. The Giardia mitosome is an ideal model for studying the limits of mitochondrial reductive evolution and, because it makes cofactors that are essential for Giardia survival, is a potential therapeutic target for this important human parasite.
with a similar cleavage mechanism to MPP [6]. Like the rickettsial homologue of MPP [16], bGPP was active as a monomer, which was demonstrated by size exclusion chromatography of recombinant bGPP as well as by analysis of bGPP from a mitosome-rich fraction separated on a sucrose gradient under native conditions (Fig. 1B, C). Importantly, kinetic parameters of monomeric bGPP (V max = 0.27 mM/min; K m = 8.4 mM, Fig. S3) were comparable to those published for the heterodimeric MPP of Neurospora crassa [8]. It has recently been suggested that the T. vaginalis HPP functions as a homodimer of two identical bHPP subunits [15], so we investigated the activity of bHPP with-and without aHPP. Unlike for bGPP, no activity for bHPP alone could be detected by gel shift assay (Fig. 1D), but a small amount of activity was observed when a highly sensitive fluorometric assay was used [15] (Fig. 1E). However, the processing activity measured by this assay increased by almost two orders of magnitude when the bHPP was associated with the aHPP-like protein, indicating that-like classic MPP-the T. vaginalis HPP functions most efficiently as a heterodimer (Fig. 1E).

Mitosomal and some hydrogenosomal targeting presequences lack distal positively charged residues
To further investigate the structure-function relationships of the GPP, HPP and MPP, we screened in silico the G. intestinalis and T.  vaginalis proteomes for putative mitosomal and hydrogenosomal Nterminal presequences (Tables S1 and S2), which were then analyzed for structural elements known to mediate substrate-MPP interactions. In particular, we searched for the positively charged residues proximal to the cleavage site (P 2 or P 3 ), and those which are N-terminally distal from the processing site. The distance between the proximal and distal group was defined to be at least 3 amino acid residues [18,19]. Giardia mitosomal presequences were predicted in three of nine putative mitosomal proteins (Table S2). All of these presequences possess the proximal P 2 arginine within a conserved cleavage motif [(ARV)R(F/L)(L/I)T], but the distal positively charged residues are absent (Table S2). The lengths of the Giardia mitosomal presequences that have been experimentally verified are 10, 12 and 18 amino acid residues. The majority of the in silico predicted Trichomonas hydrogenosomal presequences (147) resemble the Giardia pattern; having a length of 4 to 21 amino acid residues, possessing a P 2 arginine within a cleavage motif, and lacking the distal positively charged residues. However, we also detected 79 putative hydrogenosomal presequences, of 10 to 24 amino acids, that-like classic mitochondrial sequences-do contain distal arginines or lysines at position P 6 -P 22 .

Properties of MPP, GPP and HPP reflect the character of their respective substrates
To compare the specificities of the bGPP, a/bHPP and yeast a/ bMPP in vitro, we tested their activity on a selection of mitosomal, hydrogenosomal and mitochondrial substrates (Fig. 2). The bGPP cleaved only its own mitosomal substrates. By contrast, the a/ bHPP cleaved the hydrogenosomal presequences, and the presequences of mitosomal ferredoxin and two mitochondrial substrates. The yeast a/bMPP processed all of the mitochondrial substrates and the two mitochondrial-like hydrogenosomal substrates that possess distal positively charged residues. We also tested whether we could make chimeric peptidases using a combination of hydrogenosomal and mitochondrial subunits. Interestingly, while the yeast aMPP did not interact with the Trichomonas bHPP, Trichomonas aHPP was able to form a heterodimer with yeast bMPP. However, this heterodimer did not cleave mitochondrial or hydrogenosomal substrates under our experimental conditions (data not shown).
To gain further insights into the structure-function basis of their different substrate spectra, we modelled each of the different proteins ( Fig. 4 and Fig. S4), using the yeast MPP structure as a guide [10]. For yeast MPP, the substrate is first recognized by the glycine-rich loop of aMPP [6,7] and then moved to the active site of bMPP which interacts with the substrate cleavage motif including the proximal arginine. The distal positive residues of the presequence help to stabilize the substrate-MPP complex by binding to negatively charged residues within the large polar cavity formed by the a/bMPP subunits [10]. The part of the substratebinding cavity formed by bMPP thus displays an evenly distributed negative charge to accommodate both proximal and distal positively charged residues of mitochondrial presequences. The aMPP interacts only with the distal positive residues of longer (.20 amino acid residues) mitochondrial presequences [9,10].
As the bGPP functions as a monomer we predict that its substrates, including the proximal arginine, interact directly with the negatively charged region of its catalytic site (Fig. 4). The rest of the predicted bGPP cavity is, unlike bMPP, positively charged, although its predicted overall fold structure still resembles that of bMPP (Fig. S4). The difference in bGPP charge distribution is compatible with the absence of distal positively charged residues in the mitosomal presequences, and, along with the absence of an aMPP-like subunit, may explain the inability of bGPP to process mitochondrial-type presequences. The simplicity of GPP is consistent with the highly reduced function of mitosomes and likely reflects (i) the paucity of proteins that are targeted to this organelle when compared with mitochondria and (ii) lack of Nterminal cleavable presequences in the majority of mitosomal proteins, including bGPP itself. As shown above (Table S2), only nine mitosomal proteins have been identified so far and these are involved either in organelle biogenesis (Gipam18, GiHsp70, GiCpn60, GPP) or the formation of Fe-S clusters (GiiscS, GiiscU, GiiscA, Gigrx, Gifdx), which is currently the only known mitosomal function for G. intestinalis. Of these, seven are targeted to mitosomes in the absence of a detectable N-terminal targeting signal and thus function independently of GPP. Other than these, no other homologues of mitochondrial proteins have so far been identified in the genome of G. intestinalis [13].
The T. vaginalis HPP represents an intermediate stage between GPP and MPP in terms of charge distribution and enzymatic activity. Thus, it can process presequences with-or without distal positive residues, but can only cleave the shorter mitochondrial presequences (Fig. 2). The presence of mitochondrial type presequences on hydrogenosomal proteins is consistent with the retention of the aHPP, which is likely involved in their recognition via its glycine-rich loop and/or their docking at the cleavage site.
Our phylogenetic and functional analyses show that the Giardia GPP is a striking example of reductive evolution from a heterodimeric to a monomeric enzyme, with properties resembling the putative ancestral a-proteobacterial enzyme, rather than the highly specialized MPP heterodimer found in well characterized mitochondria. While the principal selective pressure for the evolution of the processing peptidases is probably their ability to efficiently process substrates, the differences in the properties of the substrate presequences may also reflect the mode of their translocation across the organelle membranes [20]. In mitochon- dria from species across the phylogenetic tree [21], the positive residues of N-terminal presequences are recognized by the outer membrane TOM system and then the inner membrane translocase complex TIM23 [3]. Interestingly, no receptors (Tom20, Tom 22, Tom70) or components of the translocation channel of the TOM complex (Tom40, Tom5, Tom6, Tom7) have so far been identified for G. intestinalis [13] or T. vaginalis [22]. Putative core components of the TIM23 translocase (Tim23, Tim17) as well as Pam18 involved in protein transfer to the matrix have been found in T. vaginalis, but only Pam18 was found in G. intestinalis [14]. It thus appears that reductive evolution of the organelles has dramatically affected both the processing peptidases and the protein import pathway [21], with important implications for general models of mitochondrial biosynthesis, structure and function.

Phylogenetic analysis
Complete sequences of bGPP, bHPP, and bMPP were aligned with Muscle [23] to calculate sequence identity and similarity values. MPP, GPP and HPP sequences were aligned with Muscle [23] and analysed with Gblocks [24] to remove ambiguously aligned sites. Bayesian phylogenetic analyses were conducted using P 4 (http://www.nhm.ac.uk/research-curation/projects/P4/index. html). The optimal substitution model for Bayesian analyses was identified by ProtTest [25] (WAG+Gamma), a polytomy prior [26], and one or more base composition vectors, which were free to vary during the chain under the NDCH model [17]. MCMC chains were run for 1,000,000 generations, sampling trees and parameters every 200 generations. Model parameter proposal tuning values were determined using the P 4 ''autoTune'' method. The burn-in was identified using the method of Beiko and coworkers [27]. The base composition component of the model was tested by simulation of the base composition x2 statistic [17] at each sampling point, resulting in a posterior predictive distribution [28] against which the statistic of the original data could be tested using tail-area probability. Composition vectors were successively added until adequate fitting of the observed data to the model was identified (see Fig. S1).

Preparation of recombinant proteases and substrate proteins
The bGPP (NCBI accession: XP_001707100), aHPP (XP_001276882) and bHPP (XP_001316822) subunits and their substrates were expressed with hexahistidine tags in E. coli. An a/ bHPP heterodimer was assembled from bHPP-His and nontagged aHPP subunits by incubation of lysates of E. coli expressing the respective proteins for 30 min on ice in 20 mM Tris, 20 mM NaCl (pH 8.6), 1 mM MnCl 2 . All recombinant proteins were purified by nickel column chromatography (HiTrap Chelating) under native (bGPP-His, aHPP-His, bHPP-His, and a/bHPP-His) or denaturing (substrate proteins) conditions. An a/bMPP heterodimer was prepared as published [18].

In vitro protease activity assays
The GPP reactions were carried out in 20 mM Tris (pH 8.0), 100 mM NaCl, 1mM MnCl 2 , 30 min at 37uC, the HPP reactions Predicted charge polarity distribution of the bHPP subunit and bGPP based on the known structure and charge distribution of the Saccharomyces cerevisiae bMPP subunit [10]. Red and blue colours denote negative and positive charge (65 kT/e where kT is thermal energy and e is unit charge), respectively, whereas white denote relatively non-polar regions. The yellow asterisk marks the Zn-binding region in the active site of the enzyme (shown in b). The negative charges are distributed evenly in the cavity of bMPP while in the cavity of bGPP the negative charges are concentrated mainly around the active site. The MODELLER program [29] version 9.2 was used to build 3-D models of aHPP, bHPP and bGPP. The electrostatic properties of the model were evaluated using APBS version 0.5.1 [33]. (B) Alignment of key segments where negatively charged residues of bMPP are located and known to interact with the substrate. Numbered residues are those of yeast bMPP. E 160 and D 164 make a salt bridge with substrate residue R-2 (P 2 ) and F 77 interacts with P 1 9 which is also often a F residue. H 70 -X-X-E 73 -H 74 is the conserved motif of the active site. doi:10.1371/journal.ppat.1000243.g004 in 20 mM Tris-HCl (pH 8.6), 20 mM NaCl, 2 mM MnCl 2 , 30 min at 37uC and activity of MPP was determined in 50 mM HEPES (pH 7.4), 20 mM NaCl, 1 mM MnCl 2 , 30 min at 30uC. To identify the cleavage sites, all substrates processed by the three proteases were subjected to N-terminal protein sequencing by Edman degradation. The kinetics of GPP was determined using the method published by Arretz and co-workers [8]. For determination of the activity of the HPP subunits, purified aHPP-His and bHPP-His were incubated on ice for 30 min either alone, or mixed together with 1 mM MnCl 2 . After addition of TviscU, the reaction was allowed to proceed at 37uC for 60 min. The specific activity of HPP with a fluorescent substrate based on the presequence of TvAK [Abz-MLST LAKRF AY(NO 2 )GKKDRM] (Bachem, Switzerland) was measured at 420 nm, with an excitation wavelength of 315 nm (Infinite M200, Tecan).

Size exclusion chromatography of purified GPP
A pre-calibrated Superdex 200 column was used to determine the molecular mass of E. coli produced GPP, under native conditions. Affinity purified bGPP-His in buffer of 50 mM CHES (pH 9.5), 150 mM NaCl was loaded on the column and washed (0.5 ml/min), collecting 1 ml fractions. Protein-containing fractions were assayed for bGPP activity.

Sucrose gradient centrifugation of a Giardia mitosomeenriched fraction
The molecular mass of GPP expressed in G. intestinalis with a hemagglutinin (HA) tag was estimated under native conditions by sucrose gradient centrifugation [15]. The mitosome-enriched fraction was isolated from a G. intestinalis homogenate using a published method [14]. The proteins in the mitosomal-enriched fraction were then separated on a calibrated sucrose gradient [15]. Fractions were analysed by immunoblot using anti-HA antibodies. Bands visualized by alkaline phosphatase were quantified by densitometry (GS-800 Calibrated Densitometer, BioRad).

Hydrogenosomal and mitosomal presequence identification
An application based on the NetBeans Platform (http:// platform.netbeans.org) was developed to search for proteins containing N-terminal hydrogenosomal and mitosomal presequences in the predicted T. vaginalis (http://www.trichdb.org/ trichdb/) and G. intestinalis (http://www.giardiadb.org/giardiadb/) proteomes, respectively. Hydrogenosmal presequences were predicted based on two main parameters extracted from 21 known hydrogenosomal presequences: (i) the cleavage site motif, specified as RXF/(ILFSAGQ) or R(FNESG)/(ILFSAGQ) (the slash indicates the cleavage site and brackets mean one residue position), and the presequence start motif defined as ML(STACGR) or MTL or MSL. In addition, tryptophan was forbidden from the presequence, the maximum presequence length was optimized to 25 residues. Any presequences with overall negative charges were excluded (the approximate presequence charge at pH7 was counted according to the Henderson-Hasselbalch equation using the following pKa values: N-terminus 8.0, lysine 10.0, arginine 12.0, histidine 6.5, glutamic acid 4.4, aspartic acid 4.4, tyrosine 10.0, and cysteine 8.5). The G. intestinalis proteome was searched for N-terminal presequences based on three experimentally verified mitosomal presequences of known mitosomal proteins [13] (Table S2). The parameters defined for the search were as follows: the cleavage site motif was defined as R(FS)/(IL)T, the presequence start motif as M(SLT), the maximum presequence length was set up to 20 residues, tryptophan was forbidden from the presequence. A search using parameters for prediction of hydrogenosomal presequences did not reveal additional mitosomal protein candidates.

Protein structure prediction
The MODELLER program [29] version 9.2 was used to build 3-D models of aHPP, bHPP and bGPP. Alignments of the bGPP and bHPP with the bMPP (pdbid 1HR6) [10] and of the aHPP with the aMPP (pdbid 1HR6) [10] were carried out using the PROBCONS web service [30] and manually edited. The quality of the final model was checked using the ProCheck [31] and WhatCheck [32] programs. The electrostatic properties of the model were evaluated using APBS version 0.5.1 [33]. Figure S1 Bayesian model composition fit to the data assessed by posterior predictive simulation. Bars show the posterior distribution of x2 for the homogeneous composition model (red) and the heterogeneous composition (NDCH) model with 10 composition vectors (green) in comparison to the statistic from the observed data. The simulated data for the NDCH model include the x2 statistic from the observed data whereas the simulated data from the homogeneous model do not, the NDCH model thus provides a much better fit to the data. The original x2 statistic for the data was 1292. In the simulations from the homogeneous analysis, this statistic ranged between 617 and 933 (mean = 763), while in the heterogeneous analysis (10 composition vectors) the statistic ranged between 877 and 1487 (mean = 1132 ). Homology models of bHPP and bGPP were built using the known structure of Saccharomyces cerevisiae bMPP, aHPP was modelled using S. cerevisiae aMPP. b-sheets are shown in yellow, a-helices in red, loops in grey. The glycine-rich loop of the a subunits and the zinc-biding motif of b subunits are highlighted in green. The MODELLER program [29] version 9.2 was used to build 3-D models of aHPP, bHPP and bGPP. The PROCHECK program version 3.5.4 was used to verify the validity of the model and gave a overall G-factor value of 20.12, which is well above 20.5; values below 20.5 indicates unusual structures [34]. Secondary structure prediction with PSIPRED [35] was also consistent with the modelled structure, recovering all five betasheets and the majority of alpha-helices (12 of 18