Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India
1 To whom correspondence should be addressed. e-mail: asharma{at}icgeb.res.in
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Abstract |
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Keywords: peptidomimics/proteinprotein interactions/PxxP motifs/SH3 modules/signal transduction
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Introduction |
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Proteinprotein interactions underpin most molecular cross-talk in cells and these interactions can occur over short regions, often <10 amino acids in length (Cohen et al., 1995; Kay et al., 2000
; Mayer, 2001
). Aside from a primary biochemical role, many proteins have additional functional attributes that are utilized in networking with cellular proteins. To define the numerous proteinprotein interactions within a cell, researchers still rely on laborious laboratory techniques such as yeast 2-hybrid systems, c-DNA expression library screening and co-immunoprecipitation (Cohen et al., 1995
; Sudol, 1998
; Kay et al., 2000
; Mayer, 2001
). Interactions mediated by modules such as the Src homology (SH) 2 and 3 domains, WW domains, post-synaptic density/disc-large/ ZOI (PDZ) domains and Eps 15 homology domains (EH domains) display a typical mode of interaction by recognizing a linear region of 39 amino acids (Sudol, 1998
; Kay et al., 2000
; Mayer, 2001
). The amino acid proline attains paramount importance in many proteinprotein interactions (Sudol, 1998
; Kay et al., 2000
; Mayer, 2001
). SH3 modules along with several other domains prefer ligand sequences that are proline-rich. SH3 domains are
60 residue modules which often occur in signaling and cytoskeletal proteins (Dalgarno et al., 1997
; Sudol, 1998
; Kay et al., 2000
). These structurally conserved domains are ubiquitous in biological systems, including in a bacterium such as the Mycobacterium (Ponting et al., 1999
; Feese et al., 2001
). Binding of SH3 domains to simple peptides folded in to proline-rich II (PPII) helix is likely to govern the formation of a large number of protein complexes (Tong et al., 2002
). Most proteins known to interact with SH3 domains contain at least one copy of the motif PxxP. SH3 domains bind to sequences that adopt a left-handed PPII helical structure in which the two invariant proline residues are found on the same face of the peptide and participate in hydrophobic interactions (Feng et al., 1994
; Lim et al., 1994
). Recent data suggest that SH3 domains can also interact with non-PXXP motifs (Kang et al., 2000
; Kami et al., 2002
), although the binding sites for non-PxxP-containing peptides and for the classical PxxP motifs do not overlap (Kami et al., 2002
).
Analysis of phage display libraries have demonstrated that individual SH3 domains have distinct specificities for potential ligands and can discern subtle differences in the ligand primary structure (Sparks et al., 1996). Much of this specificity comes from amino acids flanking the core PxxP motif (Sparks et al., 1996
). The role of PxxP-mediated proteinprotein interactions in a multitude of cellular processes is therefore central. We reasoned that dissecting the proteomes for the presence of PxxP sequences would open new vistas in understanding sets of proteinprotein interactions and signaling pathways. We therefore performed extensive in silico sieving of the complete proteomes of two pathogenic organisms, P.falciparum and Mycobacterium tuberculosis, along with the proteomes of Schizosaccharomyces pombe, Mus musculus and Homo sapiens for the prevalence of PxxP motifs. We defined a set of PxxP motifs that have been experimentally verified to bind to SH3 domains (Cesareni et al., 2002
; Tong et al., 2002
) and screened these five proteomes. We used two strategies to identify putative SH3 ligands in these proteomes: (a) screening with a consensus motif and (b) screening with specific motifs. Our results indicate an abundance of class I and class II PxxP motifs in the proteomes of two major pathogenic organisms and three higher eukaryotic organisms. Our analyses have also identified shared motifs in the proteomes of M.tuberculosis and P.falciparum which can potentially be used for the development of peptidomimics against both these organisms. We propose a novel strategy of drug target selection where multiple proteins in the cell can be simultaneously inhibited using peptidomimics that target common PxxP motifs. In line with this strategy, we have identified several motifs that are highly conserved in the cytoplasmic (the erythrocyte cytoplasm) domains of P.falciparum variant surface antigens (also called P.falciparum erythrocyte membrane proteins or PfEMPs). These PxxP motifs are absent from the human proteome. The PfEMP1 family proteins undergo antigenic variation in P.falciparum and are crucially responsible for the cytoadherence associated mortality in P.falciparum malaria (Smith et al., 1995
; Su et al., 1995
). The development of peptidomimics that may disrupt the normal functioning of PfEMPs can provide a new focus for the development of anti-malarials that target cytoadherence of P.falciparum.
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Methods |
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Results |
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We identified all proteins with at least one occurrence of the PxxP motif in the proteomes of human, mouse, S.pombe, M.tuberculosis and P.falciparum. The resulting motifs were delineated into classes and subclasses keeping in view published nomenclatures (Rickles et al., 1995; Sudol, 1998
; Cesareni et al., 2002
; Tong et al., 2002
). A large fraction of the diversity observed in SH3I domains is represented in the SH3 repertoire of Saccharomyces cerevisiae (Cesareni et al., 2002
; Tong et al., 2002
). Therefore, we selected the SH3 domain containing proteins of S.cerevisiae and their PxxP motif recognition specificities as a guide to analyze the five proteomes considered. We followed the PxxP motif nomenclature published recently (Cesareni et al., 2002
) and classified the PxxP sequences into three major classes: 1K, 2K and 1@. All other atypical PxxP motifs are grouped under Class X. The various subclasses have been defined based on highly probable consensus motifs which have also been experimentally verified for binding to different SH3 domains (Kay et al., 2000
; Mayer, 2001
; Cesareni et al., 2002
; Tong et al., 2002
). We used the consensus sequences for various classes and subclasses as queries and filtered out the hits for each proteome. Using this motif-based proteome scanning approach, we identified all PxxP motifs in the annotated and predicted proteins (Figure 2a). The number of PXXP motifs in each proteome was plotted as a percentage of total number of proteins (Figure 2a). The P.falciparum proteome revealed the lowest fraction of PxxP motifs amongst the proteomes (
25.9%). The M.tuberculosis proteome revealed a relatively high fraction of proteins contained the PxxP motif (
58.4%). Surprisingly, the S.pombe proteome contained the maximum fraction of proteins with PxxP motifs (
70.2%). Amongst the mammalian species, both human and mouse proteomes had a comparable fraction of PxxP motifs (
65.2 and
61.8%, respectively). These data reveal the abundance of PXXP motifs in proteins from evolutionary distant species.
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Omnipresence of PxxP-containing proteins
We next classified the PxxP-containing proteins from each proteome into categories based on the likely cellular localization of each protein (Table II) and by gene ontology (Table III). The data indicate that PxxP motifs are prevalent in cytoplasmic, nuclear and surface proteins. We found an abundance of PxxP motifs in various gene ontology groups of proteins including enzymes, cytoskeletal proteins, nucleic acid-binding proteins, transport proteins, splicing factors, metal-binding proteins and ribosomal proteins (Table III). This wide occurrence across diverse functional classes in the five proteomes indicates evolutionary conservation of proteinprotein networks centered on PxxP sequences. The prevalence of PxxP motifs in biological systems is consistent with the ubiquitous nature of SH3 domains and together the SH3-PxxP interactions are central in a diverse array of cellular communication processes (Sudol, 1998; Kay et al., 2000
; Mayer, 2001
).
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The number of major classes of PxxP motifs in each organism was plotted as a percentage of total number of PxxP motifs in that respective organism (Figure 2b and Table IV). Analysis of these data reveals that the majority of the motifs can be classified either as a Class I (1K) or a Class II (2K) motif (Mayer, 2001; Cesareni et al., 2002
). The Class I PxxP motifs are defined as sequences that fit the amino acid stretch +xxPxxP (where + is a basic residue and x is any amino acid). Class II PxxP motifs are sequences which match PxxPx+ and 1@ represents sequences that fit the motif Px@xxPxxP (where @ is an aromatic).
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Subtle differences in binding affinity and specificity in PxxPSH3 interactions have been attributed to residues outside of the PxxP motifs (Cesareni et al., 2002). Several studies have used phage display libraries to identify the specific ligand of different SH3 domains (Rickles et al., 1994
, 1995
; Sparks et al., 1996
). The optimal ligand preference for each SH3 domain varies around the PxxP core (Table IV). We reasoned that scanning the five proteomes with greater stringency would yield information on the appropriate SH3 receptors. The subclass delineation was based on definition of PxxP-containing sequences (Cesareni et al., 2002
). Analysis of the data (Table IV) clearly indicates a preference for a subclass of motifs within each major PxxP class, suggesting reliance on proteinprotein interactions based around some SH3 domain- containing proteins. We also plotted the number of different 1K ligands in the five proteomes considered as a percentage of the total number of proteins containing PxxP motifs (Figure 3a). Amongst the Class I motifs (Table IV), the PxxP ligands with preference for SH3 domain-containing proteins Rvs167, Pex13, Hck and SHO1 are highly represented. This distribution is likely to be of biological significance. The yeast Rvs167 protein plays critical roles in actin cytoskeleton organization and endocytosis (Breton et al., 2001
). It interacts with myosin and participates in vesicle traffic and maintenance of cell integrity (Breton et al., 2001
). The yeast Pex13 encodes for an SH3 domain-containing peroxisomal membrane protein required for the import of proteins into peroxisomes (Winkler et al., 2002
). Hck is a prototypical member of the tyrosine kinase family and the yeast SHO1 protein is a membrane-bound osmosensor that is involved in stress activation (Winkler et al., 2002
). Together, these data suggest that a substantial number of proteins in each of the five proteomes may use SH3PxxP interactions to drive essential cellular processes of signal transduction, cytoskeleton organization, protein transport and osmoregulation.
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The distribution of 1@ subclasses is shown in Figure 3d. The highest occurrences are observed for spectrin-binding motifs followed by those for myosin and the tyrosine kinase protein Abl. The tyrosine kinase Abl participates in several processes including in the apoptotic response of cells to DNA damage (Yoshida et al., 2002) and is specific for atypical PxxP motifs. The SH3 domain of spectrin is implicated in a variety of myosin- and spectrin-based cytoskeleton organization (Ziemnicka-Kotula et al., 1998
; Geli et al., 2000
).
Conformations of both proline (backbone angle
65°) and the flanking residues are limited because of the bulk of the N-substituent in proline (MacArthur and Thornton, 1991
). Therefore, polyproline sequences tend to adopt the PPII helix conformation which is generally solvent exposed and amphipathic in globular proteins (Stapley and Creamer, 1999
). We evaluated 4531 entries (which contained PxxP motifs) from the protein structure database (PDB) and found that 1115 entries have PxxP motifs which are 50% solvent exposed, 370 entries have PxxP motifs which are 70% solvent exposed and 36 entries have PxxP motifs which are 90% solvent exposed (the programs MODELLER and PROCHECK were used to detect residue accessibility of PxxP motifs). These solvent-exposed PxxP motifs have the potential to interact with SH3 modules (some examples of accessible Class I and II motifs are shown in Figure 4). Indeed, our recent crystal structure analysis of Pfg27 from P.falciparum revealed a striking distribution of exposed PxxP motifs in the protein, which readily interact with SH3 modules (Sharma et al., 2003
).
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The development of peptidomimics that specifically inhibit interaction of PxxP motifs with their cognate receptors is feasible given the unique structure of the proline residue (Nguyen et al., 1998; Oneyama et al., 2002
, 2003
). Therefore, we surveyed the total number of PxxP motifs that are unique to either P.falciparum or M.tuberculosis (Table V) with the aim of identifying sequences that could serve as leads for the development of peptide-based inhibitors. Indeed, there are a large number of unique PxxP motifs in both of these organisms. With the aim of identifying lead sequences for the design of drugs against malaria and tuberculosis, we found PxxP motifs that are common to both P.falciparum and M.tuberculosis but are absent from human proteins. The following sequences are not present in humans but are common to M.tuberculosis and P.falciparum: PAPAAPSS (found in PPE family protein and PfEMP), IGPNCPGI (found in succinyl-CoA synthetases) and SSPNTPGL (found in dihydroorotate dehydrogenases). Experimental verification of the biological necessity of these motifs may pave the way for their inclusion in drug development efforts against malaria and tuberculosis.
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Discussion |
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Close to 65% of the P.falciparum proteome is unannotated and >3000 proteins remain hypothetical (Florens et al., 2002). Here, we have used a simple PxxP motif search to predict putative proteinprotein interactions in P.falciparum and other organisms. We had previously highlighted the interaction of P.falciparum gametocyte protein Pfg27 with different SH3 domains and discovered a striking structural display of PxxP motifs in the Pfg27 underbody (Sharma et al., 2003
). Our analyses suggest that the distribution of PxxP motifs in P.falciparum proteins is therefore likely to be of significance in understanding parasite biology.
Proteinprotein interaction networks have been studied in S.cerevisiae with respect to SH3 modules. Tong et al. showed that using a set of 18 SH3 modules in 2-hybrid analysis there are 233 interactions involving 145 PxxP-containing proteins (Tong et al., 2002). Using the technique of phage display, they observed 394 SH3PxxP interactions from 206 proteins. These data imply that a vast number of proteinprotein interactions are mediated using the SH3PxxP cross-talk.
Our comparative distribution analyses of PxxP motifs across various proteomes, which include the proteomes of infectious organisms such as M.tuberculosis and P.falciparum, can underpin ongoing efforts to dissect and delineate the plethora of signaling pathways in these organisms. It also provides a basis for further biological characterization of PxxP motifs that can be utilized for the design of peptidomimics. Specific sequence motifs offer a platform for the design of peptidomimics that specifically target multiple pathways in cells. This novel strategy may provide a new focus for the development of anti-tuberculosis and anti-malarial agents.
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Acknowledgements |
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References |
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Received October 22, 2003; revised January 6, 2004; accepted February 2, 2004 Edited by Alan Fersht