Comparative Structure Modeling of Metal Regulatory Transcriptional Factor ; MTF-1

Metallothionein is a heavy metal binding cystein rich protein, of which is mediated by the binding of heavy metal transcription factor (MTF-1), to its metal response elements (MREs). But its exact signaling pathway and the structures of interacting molecules are not very clear. In order to predict the structure of MTF-1, modeller 9v1 mediated alignment of National Center for Biotechnology Information (NCBI) sequence CAC14279, containing 791 amino acids, with non-redundant (Protein Data Bank) PDB sequences was performed. Since the result was not supportive for the full length modeling of the sequence, best aligning region falling from 111 residue to 284 residue was selected and modeled. The rest was submitted for Protein Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) and the GeneThreader. Based on the results, 284 residue to 360 residue region was modeled and the two structures were matched by chimera to produce a single structure accounting for 31.47% of the total sequence length of MTF-1. The rest of the sequence accounting for 68.53% requires more information on its folding pattern. Thus experimental approaches such as X-ray crystallography and Nuclear Magnetic Resonance (NMR) need to be employed. INTRODUCTION Metallothioneins (MTs) constitute a conserved family of cysteine-rich heavy metal binding proteins. MT is found ubiquitously in the tissues of many animal species and plays a role in the homeostasis of essential metals such as zinc and copper (Basu and Lazo, 1990). From insects to mammals, metallothionein genes are induced in response to heavy metals transcription factor (MTF-1), a zinc finger transcription factor, which binds to short DNA sequence motifs, termed metal response elements (MREs) by direct zinc binding to the MTF-1 zinc fingers (Bittel et al., 2000; Chen et al., 1999). The activation of MTF-1 is mediated by the presence of heavy metals, thus the heavy metal detoxification is controlled to a large extent by the MTF-1. In searching data bases for the sequence of MTF-1, Expressed sequence Tags (EST) sequences CAC14279 derived from adult fly heads and from schneider cells were found and cDNA encodes a protein of 791 amino acids, which is slightly larger than vertebrate MTF-1. It has 78% similarity in the zinc finger region and 27% outside it, i.e., 39% in the total protein with the vertebrates (Dalton et al., 2000; Maur et al., 1999). Experiments have shown that MTF-1 is strongly expressed in the gut of adult Drosophila and larvae (Bonneton and Wegnez, 1995) but the exact signaling system and the 1 Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Peradeniya, SriLanka. Comparative Structure Modeling of MTF-1 structures of the interacting molecules which is the limiting factor for the predicting of the signaling pathway, remains to be discovered. In this study prediction of the structure of MTF-1 was attempted using evolutionary information. MATERIALS AND METHODS For the prediction of a possible structure for the MTF-1, comparative structure modeling was attempted. Initially the sequence CAC14279, containing 791 amino acids obtained from National Center for Biotechnology Information (NCBI) was used to find a matching profile, using “pdb_95. pir” text format file containing non-redundant Protein Data Bank (PDB) sequences at 95% sequence identity in the database. Due to the template sequence and PDB sequence length mismatch, a multiple sequence alignment between the MTF-1 sequence and PDB sequences was performed using CLC Free Workshop with an open gap penalty of 10 and gap extension penalty of 1. In order to find a suitable known three-dimensional structure homologous to MTF-1, PSI-BLAST (Protein Specific Iterative Basic Local Alignment Search Tool) (Altschul et al., 1997) was performed using the engine located at NCBI and gene threading was done using the mGeneThreder (McGuffin et al., 2003; Jones, 1999). Considering the aligning region of non-redundant PDB sequences with MTF-1, section ranging from 111 residue to 284 residue was modeled using the modeler 9V1. Remaining sequences were again aligned with non-redundant PDB sequences followed by a PSI-BLAST and gene threading (Jones, 1999; McGuffin et al., 2003). Considering the results another section ranging from 260 to 340 residue was modeled using the modeler 9V1. Since there were not enough information available for modeling the rest of the sections, completed models were matched using University of California San Francisco (UCSF) Chimera (Pettersen et al., 2004; Pei et al., 2001) with the Needleman-Wunsch algorithm with a secondary structure score of 30% and a gap extension penalty of 1 to create a single structure. RESULTS AND DISCUSSION NCBI sequence CAC14279, containing 791 amino acids, alignment with “pdb_95. pir” text format file containing non-redundant PDB sequences resulted six PDB structures having the PDB identities as 1g2dC, 1g2fC, 2gliA, 1meyC, 1tf3A, and 1tf6A. (Wolfe et al., 2001; Nolte et al., 1998; Foster et al., 1997; Kim and Burg, 1996; Pavletich et al., 1993;) (Figure 1).