1fOO
3.2.4 Secondary structure features of proteins similar to Pc96
more similar to Pyy78 than Pc96. The putative T cell epitopes, predicted in Section 2.1.2, are indicated in the alignments, along with the casein kinase II phosphorylation site, shared between, Pf403 and Pc96.
250 bp region of high similarity. The plots generated over theT cell epitopes show the nature of the secondary structure, and indicate the similarities of the sequences containing the conserved epitopes on Pf403 and Pyy178.
3.2.5 Motif and pattern recognition using PROSITE and domain analysis using 3D-PSSM
Table 3.7 shows the identification of PROSITE profiles for the proteins used in this study. These . proteins contain regions of sequence similarity to that ofPc96.The N-glycosylationsites, protein kinase C phosphorylation sites, casein kinase IT phosphorylations sites and tyrosine kinase phosphorylation sites, are detected quite frequently within the sequences of these proteins. The Leucine zipper pattern detected in all proteins except Pyy78,has previously been shown to play a role in gene regulation. Although false hits have been identified in protein sequences known to lack this motif, verification is usually obtained by the identification of DNA binding motifs. In Pf403 there are three bipartite nuclear targeting sequences, increasing the likelihood of Pf403 playing a role in DNA binding or gene regulation. Although it may be difficult to draw any structural or functional conclusions about these proteins, these consensus sequences comprising of detected block motifs in the PROSITE database can be used for comparative analysis of 3D structure, and the possible assignment of function for certain domains.
Table 3.7. The presence and number of motif and sequence patterns identified using PROSITE for Pc96, Pf403, Pyy178 and Pyy84.
PRO SITE pattern entry Pc96 Pf403 Pyy78 Pyy84
.N-g1ycosylation site 20 38 27 10
cAMP and cGMP-dependent protein kinase I 6 2 2
phosphorylation site
Protein kinase C phosphorylation site 9 16 26 6
Casein Kinase II phosphorylation site 24 49 35 14
Tyrosine kinase phosphorylation site 3 18 7 2
N-myristoylation site 2 6 8 8
Leucine Zipper pattern 2 1 1
Sulfonation 12 25 14 1
NUCLEAR Bipartite nuclear targeting sequence 3
proteins under study (Pf403, Pc96, Pyy178 and Pyy84). This particular program recognises certain combinations and positions of signature sequences and motif patterns from the sequence and compares this to a database with a collection of known 3D structures. The statistical relevance of the matches were reported as E values, reflecting the given probability of the domain recognition pattern occurring by chance, based upon the initial alignments in the PSSM algorithm. In general, E values below 0.1 are considered significant, and matches with values below 1 can still be investigated.
In an attempt to recognise specific structural and functional domains, overlapping regions of Pc96 and homologous proteins Pyyl78 and Pyy84 were submitted for fold recognition at the 3DPSSM site. This provided clues as to the identity of these regions within the proteins.
Analysis of Pc96 (Table 2.8) shows the identification of a region highly similar to the botulinum neutoxin,and the yeast t-snare protein (ssol), a membrane protein. The Pyy178 protein sequence was split into three regions, identifying the colicin la domain in the first, and the botulinum neurotoxin in the second and third. The hydrolase domain was again found, similar to that of phospholipase c beta and a region homologous to a hydrolase activator, proteasome component y7, was identified. Pyy84 showed homology to Botulinum neurotoxin and interferon-induced guanylate-binding protein I, a DNA binding protein.
These features compare to that of Pf403, indicating a similarity in the 3D structure of Pc96 as well as the identified regions of sequence similarity. As can be seen on Table 3.9, a significant match with Colicin la was obtained located in the first 500 residues of Pf403. This indicates the possible role of Pf403 in biological membrane association as colicin la is involved in ion channel forming in membranes and cell surfaces (Stroud et al., 1998). The interferon-induced guanylate-binding protein 1, may also indicate DNA-binding properties. The domain that showed the highest degree of structure recognition was the Botulinum neurotoxin catalytic domain. This protein is a zincin metalloprotease, and is also involved in membrane association.
It is interesting to see the recognition of this domain, and that of colicin IA, in two specific regions of Pf403. The hydrolase domain found in angiostatin, was recognised in the last 500 residues,along with translation initiation factor eIFla,a DNA-binding protein. Another putative hydrolase domain was detected.in the first series of residues screened, similar to that of phospholipase c beta.
Table 3.8.3D structure comparisons using 3DPSSM and identification of putative domains in Pc96, Pyy178 and Pyy84.
E value of Protein Protein Name Description Organism
region Pc96(l-480)
0.0259 Botulinum neurotoxin serotypeA Clostridium neurotoxins, Clostridium catalytic domain-Zincin-like botulinum metalloprotease
0.12 yeast t-snare protein ssol membrane protein Yeast
Pc96(481-942)
0.0254 Botulinum neurotoxin serotypeA Clostridium neurotoxins, Clostridium catalytic domain - Zincin-like botulinum metalloprotease
0.0597 GST-alpha-Na,K-ATPase crystal structure of the ankyrln binding domain of2 alpha- Na,K-atpase as a fusion protein with glutathione3 s-transferase - ankyrin binding.
Pyy178(1-488)
0.129 Colicin la Membrane and cell surface Enteric bacteria
protein - ion channel forming
0.329 Interferon-induced guanylate-binding c-terminal domain - binding of Homo sapiens
protein 1 guanine nucleotides
0.489 phospholipase c beta c-terminal- hydrolase enzyme Pyy 178(488-977)
0.000771 Botulinum neurotoxin serotypeA Clostridium neurotoxins, Clostridium catalyticdomain - Zincin-like botulinum metalloprotease
0.0487 yeast t-snareprotein sso l membrane protein Yeast
Pyy178(977-l523)
0.00312 Botulinum neurotoxin serotypeA Clostridium neurotoxins, Clostridium catalytic domain-Zincin-like botulinum
metalloprotease .
0.052 proteasome component y7 hydrolase/hydrolase activator
Pyy84 (1-713)
0.171 Botulinum neurotoxin serotypeA Clostridium neurotoxins, Clostridium catalytic domain - Zincin-like botulinum metalloprotease
0.642 Interferon-induced guanylate-binding c-termina1 domain -bindin~of Homo sapiens
protem 1 guanine nucleotides- signa ling
protem
Table 3.9. 3D structure comparisons using 3DPSSM and identification of putative domains in Pf403 (Plasmodium falciparumi.
E value (region ofPf403) Protein name Description Organism
Pf403(1- 780)
·0.042 Colicin la Membrane and cell surface Enteric bacteria
protein - ion channel forming
0.326 lnterferon- induced c-terminal domain - binding of Homosapiens guanylate-binding guanine nucleotides-signalling
protein I protein
0.445 phospholipase c beta c-terminal - hydrolase enzyme Pf403(781-1560)
0.0000692 Botulinum neurotoxin Clostridium neurotoxins, catalytic Clostridium
serotypeA domain - Zincin-like botulinum
metalloprotease Pf403(1561-2340
0.0605 Colicin la Membrane and cell surface Enteric bacteria
protein - ion channel forming
0.544 lnterferon- induced c-terminal domain - binding of Homosapiens guanylate-binding guanine nucleotides
protein 1 Pf403(2341-3120)
0.0000688 Botulinum neurotoxin Clostridium neurotoxins, catalytic Clostridium
serotypeA domain - Zincin-like botulinum
metalloprotease Pf403(3121-3394)
0.254 Angiostatin hydrolase Homo sapiens
0.265 Translation initiation Nucleic acid binding - Cold shock Homo sapiens factor eIF1a DNA-binding domain
These results together were used to construct a map of Pf403 and related proteins, according to regions where putative domains of known 3D structure were identified. Figure 2.8 shows Pf403, Pyy178, Pyy84 and Pc96, with the most significant matches obtained from the 3DPSSM analysis.
Both Pyy84 and Pyy178, two separateP. yoelii yoeliiproteins showed homology to two separate regions in Pf403, sharing similar comparisons to the 3D structures in these domains.
0 500 1000 1500 2000 2500 3000 3500
I I
A I C1cii I DstiaaS
H
C1cii DSliaaSH
C1l«l1I Om3ll1 I
B DSliaaS I C1t.u5 I C
~ DSliaa3
CWnlb I
C1cii DSliaaS I 0
Figure 3.8 Diagram showing domain regions identified by 3DPSSM analysis. Proteins A -
Pf403; B - Pyy84;C - Pc96;D - Pyy178. Structures identified by 3DPSSM shown on the diagram are: Clcii (Colicin la) - shaded orange; D3btaa3 (Botulinum neurotoxin serotype A) - shaded red;C140a (Angiostatin hydrolase);Clbg5 (GST-alpha-Na,K-ATPase) and Clfntb (proteasome component y7 hydrolase activator); Didg3al (Interferon-induced guanylate-binding protein 1) _ shaded blue.
Due to the fact that two regions in Pf403, and regions in Pc96, Pyy84 and Pyy178 showed a similar 3D structure to that of Botulinum neurotoxin serotype A, with comparatively high estimations of statistical similarities, the 3D structure viewing software Rasmol was used to generate structures of the protein (Figure 3.9). Both the backbone and ribbon structures were shown, to emphasise features of the shape, and the positioning of helices and sheets making up the protein.
Figure 3.9. The 3 dimensional structures of the Botulinum neurotoxin domain, identified by 3DPSSM. Images are shown as stereograms for 3D visualisation. Panel A shows the ribbon structure and panel B, the backbone line structure.