1. H1 H2 LB LE H3H4H5H6 Bioinformatics study of Aquaporins in Dicot and Monocot plants. Neel Duti Prabh, Ravi Kumar Verma, Ramasubbu Sankararamakrishnan* Department of Biological Sciences and Bio-Engineering, Indian Institute of Technology Kanpur, Kanpur – 208016, India ABSTRACT: The major intrinsic proteins (MIP) form a family of channel proteins which are known to facilitate passive transfer of water and other small polar molecules through biological membranes. As MIPs are found in virtually every organism their function seems to be vital for life [1]. Interestingly, compared with other life forms plants seem to have greater number of MIP isoforms, in case of model plant Populus trichocarpa 55 MIPs have been identified [2]. In the last decade many plant genomes have been sequenced which opened up the possibility of analysing all the MIP members in plants. Based on phylogenetic analysis, the MIP members from vascular plants can be divided into mainly five sub-groups named: PIPs (Plasma membrane intrinsic proteins), NIPs (NOD26-like intrinsic proteins), SIPs (Small and basic intrinsic proteins), TIPs (Tonoplast intrinsic proteins) and XIPs (X-intrinsic proteins) [2]. The fifth sub-group XIPs has been found in dicot plants but no XIP homolog was found in monocots [3]. The absence of XIPs in monocots prompted us to compare the structural features of the other sub-groups between monocot and dicot MIP members. So far no comprehensive study has been done which collates aquaporin data from different plant species in monocot and dicot sub-class and explicitly compares structural features that determine substrate specificity between the corresponding sub-groups from each sub-class or different sub-group within the same sub-class. In the present study, 237 new plant MIP members have been identified using BLAST/tBLASTn search. Using Modeller software homology modelling of all the members was carried out [2]. Additional plant MIP members extracted from MIPModDB database were added to the data set [4]. Total 558 (274 from dicots and 284 from monocots) modelled aquaporin structures are analysed for variation as well as conservation of structural features. INTRODUCTION: Major intrinsic proteins (MIP) form an ancient superfamily of channel proteins that are engaged in passive transport of water, glycerol and other small neutral molecules. They have an hour-glass-like helix-bundle structure that is conserved from archaea to higher organisms. This structure consists of six trans-membrane helices and two half- helices that in the native structure come together to form the seventh pseudo transmembrane helix (Fig. 1) [2]. REFERENCES: 1. Danielson et al., Adv. Exp. Med. Biol 2010. 679, 19-31 2. Bansal and Sankararamakrishnan, BMC Structural Biology2007, 7:27 3. Gupta and Sankararamakrishnan, BMC Plant Biology2009, 9:134 4. Gupta and Sankararamakrishnan, Nucleic Acids Research, 2012, Vol. 40 ACKNOWLEDGEMENT: Neel Duti Prabh thanks IIT Kanpur for financial assistance. METHODS:  Nucleotide collection (nr/nt) database at the National Centre for Biotechnology Information (NCBI) was searched for MIP genes using TBLASTN. Sequences of MIPs representing the four subfamilies from Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Zea mays were used as query sequences. Additionally, XIPs from Populus were also considered as query sequences.  Corresponding protein sequence for all the hits were obtained directly from NCBI or after translation using Softberry- BESTORF software. Truncated proteins and sequences already available in MIPModDB database were removed.  Homology models for 237 sequences were constructed using MODELLER 9.11 software.  SCWRL 3 was used for side chain refinement.  GROMACS package was used for minimization.  ClustalO was used for multiple sequence alignment.  Phylogenetic trees were constructed using Neighbor Joining (NJ) Method. Selectivity Filter:  Residues forming the Ar/R selectivity filter in each subfamily were analyzed and compared between monocots and dicots in Table 1.  No major differences were found while comparing a given sub-group between monocot and dicot plants.  Few TIPs from monocots have a glutamine (Q) at the H2 position which is not observed in dicots.  Between the sub-groups, selectivity filters show considerable variation. Loops and termini:  Loops and termini regions do not exhibit any major difference between monocots and dicots.  Loops connecting the transmembrane segments and terminal regions show variations in terms of length and presence of charged residues between different sub- groups. MIPs form two distinct families in mammals: aquaporins and aquaglyceroporins based on solute preferences. In plants, they form at least five sub-groups namely PIPs (Plasma membrane intrinsic proteins), NIPs (NOD26-like intrinsic proteins), SIPs (Small and basic intrinsic proteins), TIPs (Tonoplast intrinsic proteins) and XIPs (X- intrinsic protein)[2]. Flowering plants (Angiosperms) are divided into two sub-classes: monocotyledons (monocot) and dicotyledons (dicot). Both sub-classes have high number of MIP family members, but so far XIPs have only been found in dicot plants [3]. CONCLUSION:  No major differences are observed between monocot and dicot MIPs, with the exception that XIPs are absent from monocots.  Each subfamily has its distinct range of selectivity filters.  Functionally significant loops Loop-B and Loop-E show conservation both in terms of length and residues within each subfamily across all plants.  A strongly conserved proline residue from Loop-E in TIPs and NIPs might have some functional significance. Table 1: Aromatic/arginine selectivity filter residues in each subfamily of monocot and dicot plants. H2 from helix – 2, H5 from helix – 5, LE1 and LE2 from loop–E Figure 3: Average length of termini and Loop regions in each subfamily of dicot and monocot MIPs. D – dicot, M - monocot PIPs TIPs SIPs NIPs XIPs DICOT PIPs TIPs SIPs NIPs MONOCOT AIM:  To investigate possible differences between MIPs from dicots and monocots.  To identify distinguishing features within the different plant MIP subfamilies. Figure 1a: Protein topology diagram of an MIP channel showing transmembrane helices and NPA motif for Loop-B and Loop-E. 1b: AQP1 aquaporin water channel [PDB Id 1J4N] monomer’s 3 – dimensional representation 1c: Superimposed selectivity filter residues of water specific AQP1 and glycerol specific GlpF [PDB Id 1FX8]. Figure 2: Phylogenetic trees for 284 monocot MIP sequences (2a) and 271 dicot MIP sequences (2b). PIPs – red, TIPs – green. NIPs – brown, XIPs – pink and TIPs – blue. AQP 1, GlpF and AqpM (PDB Id 2F2B] used as reference sequences. 2b 2a 4b 4a Figure 4: Sequence logos for Loop-B and Loop-E for each subfamily of dicot and monocot MIPs. 1a 1b 1c RESULTS and DISCUSSION:  237 new plant MIP sequences were found and modeled. In addition to these newly identified sequences, we also considered all the plant MIPs from MIPModDB database[4].  Total 558 sequences were analyzed (274 - dicots and 284- monocots).  Phylogenetic analysis showed, that monocots and dicots shared 4 sub-groups : NIPs, PIPs, SIPs, TIPs.  A fifth subfamily XIPs is found only in dicots.  PIPs are most abundant in number followed by TIPs and NIPs. Figure 5a: Superimposed residues of selectivity filters from AQP1 and a modeled NIP. 5b: Superimposition of selectivity filter residues from GlpF and the same modeled NIP. 5c: Residues showing the conserved proline (in green) from Loop-E (marked with yellow arrow) near the selectivity filter residues (in red) from the modeled NIP. Loop-B and Loop-E:  Lengths of functionally important loops Loop-B and Loop-E of all the sub-groups are strongly conserved between the monocots and dicots.  Sequence logos of Loop B and Loop E regions are presented in Fig. 4 for different subgroups. Residues which are uniquely conserved in a specific subgroup are indicated by arrows. 5a5b5c  A serine (S) near the conserved NPA motif is strongly conserved in both monocot and dicot PIPs. This feature is not observed in other subfamilies.  At same position, cystine (C) is conserved in dicot XIPs.  NIPs and TIPs show conservation of proline (P) residue in Loop-E as indicated by an arrow in Fig. 4a and marked in Fig 5c. It is possible that the presence of proline might be playing some role in the transport of solutes specific to these subfamilies.  SIPs have a conserved tryptophan (W) near the conserved NPA motif in Loop-E, but a conserved histidine (H) near the NPA motif of Loop-B found in everyother subfamily is missing from SIPs.