Discussion of SNAPG

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The SNAP-gamma structure obtained Danio rerio possess 4 chains of alpha helical hairpins fold without any beta strands observed (Figure 1). Each chain was interconnected with one another via hydrogen bond and non-bonded contacts (PDBSum). The helices towards N-terminal end were connected by extended loops and those towards C-terminal end tend to form a globular bundle presumably forming a cleft. Two different types of ligand interact with SNAP-gamma differently. One of the ligand types, sulfanate ion (SO4), was found to from a distinct interaction with different residues in different chain (Figure 2 & 3). Meanwhile, selenomethione (MSE) ligands were integrated into protein structure as seem to appear as modified amino acid residues (Figure 2).

Although the protein structure of SNAP-gamma was already known, the function is still yet under investigations. Protein super families identification reveals that SNAP-gamma is made of 2 domains in which both of the domains had a soluble-NFS(N-ethylmaleimide-sensitive factor)-attachment-protein (SNAP) activity. The sequence conservation against the protein super families (Figure 15) suggests that SNAP proteins may have a conserved 3D shape. Structural alignment against a representative structure in PDB databases using Dali web server shows several proteins with acknowledged protein function and domains shares structure similarity with SNAP-gamma protein (Table 1). For this study purpose, two proteins with highest statistical significance of the match (Z-value) were chosen. A vesicular transport protein sec17 found in yeast and type 4 fimbrial biogenesis protein pili in Pseudomonas aeruginosa had a Z-value of 23.3 and 12.9; and shares 23 and 14 respectively %identity with SNAP-gamma. Structure-based alignment of these structures indicates similar secondary structure and motifs (Figure 5&6). In regards of SNAP-gamma and sec17 surface properties proximity (Figure 7&8), it suggest that both of these proteins may have similar protein-protein interaction. However, the amino acid conservation were not satisfying (Figure 5) suggesting the function can not be determined based on structure alignment alone.

The SNAP-gamma hydrophobicity and electrostatic potential properties were distributed throughout majority of SNAP-gamma residues. These features may be important for examination of possible SNAP-gamma biological function. Large hydrophobic surface area, as shown by Figure 7.A, may contributes to ligands recognition and binding. Vast majority of SNAP-gamma negative electrostatic potentials (Figure 9) and cleft binding predictions (Figure 10) towards C-terminal end gave an insight of protein possibility to bind with another protein, but not DNA, at this region. The DNA enveloped by negative electrostatic potential just like SNAP-gamma suggests its implausibility to form an interaction with one another . This also supported by ProFunc results of which there was no HTH (helix-turn-helix) motif, which resembles DNA-binding protein, found in the structure. These finding agrees with study done by Tani in 2003 that characterized SNAP-gamma interactions with NSF via its extreme C-terminal region.

Gamma-Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein ( -SNAP) is capable of stabilizing a 20 S complex consisting of NSF, -SNAP, and SNAP receptors (SNAREs), but its function in vesicular transport is not fully understood (Tani et.al, 2003). According to the Gene Ontology database, SNAP gamma has been annotated into three different gene products; biological processing which are intra-Golgi vesicle mediated, membrane fusion, protein complex assembly and protein stabilization; molecular function which is protein binding and lastly cellular component which are membrane fraction and mitochondrion. Although all of these finding are still bias or only by similarity however all three gene products were linked each other (Figure 11). Determining SNAP-gamma function was rather difficult task as its existence is distinct from SNAP-alpha or SNAP-beta. Therefore structure-function approaches were used to find its function analysis. SSM (Secondary Structure Matching) results showed that 2ifuA for SNAP-gamma had a 24% identity of fold matching to 1qqeA. This was the closest match compared to structure from any other sequences (Figure 12 and Figure 5A). However, the Z-score which determines statistical significance of the match is low (3.6). This means that the similarity in structure does not determine similarity in function. Similar result was also presented by DALI server (Figure 1 and Figure 5B) with slightly different outcome as 2fi7A was another matching fold for 2ifuA. 1qqe is structure found in vesicular transport protein while 2fi7 involves in protein binding. Conserved residue domain alignment is another approach in suggesting SNAP-gamma protein function (Figure 13 and Figure 6); aligned 2ifuA, 1qqeA and 2fi7. Additional sources were also applied for SNAP-gamma function prediction including sequence motif, genomic context, cellular context and literature context. In term of sequence motif, Aromatic-di-Alanin (AdAR) repeat was found in NSF attachment protein family in which this repeat similar to that found in TPR-1 (Figure 14). TPR-1 is a member of TPR superfamily. Both genomic context and cellular context contributed additional data for this study. The SNAP-gamma protein appeared to be conserved various organisms’ genome throughout evolution including human and mouse. Mostly the genes were found in H.Sapien and M.musculus (Figure 15). Cellular context showed localization of SNAP-gamma in different tissue areas of the body (Figure 16 and 17).

Figure 1. Evolutionary analysis of the tree with radial view. Red dotted signs means bootstrap value more than 75% or 75.00

SNAP-gamma protein evolutionary tree mostly contains more than 75% boostrapvalue. Thus, red dot signs was assign to represent the branching tree which has more than 75% bootstrap value. The value was to measure the consistently data compare with the branching tree patterns. As an example: in the diagram phylogenetic tree (see Figure 3), there is 39.0 boostrap value in branching Apis melifera and Tribolium castaneum. The boostrap value is below 75%, thus it could be concluded that the degree of confidence of branching tree in the correct order is far below 95%. On the other hand, boostrap value more than 75% or 75.00 means 95% confidence that the branching tree is in the correct order. It means, if in certain branch and the organism include in this branch have bootstrap value more than 95%, nearly all the informative sequence of this group agree that it is the group.

As indicated in diagram in Figure 1, SNAP-gamma protein was evolved in eukaryotes. There is no prokaryotes organism detected in the phylogenetic tree. The SNAPG protein was detected in lower eukaryotes until higher eukaryotes. Interestingly, SNAPG protein has similar function although it evolved in different organism. By homologous gene sequence comparison, the function of SNAPG in eukaryotes is to facilitate vesicular transport between Endoplasmic Reticulum and Golgi Apparatus. Unfortunately, the exact function of this protein in each organism is not yet known.