G3p

From Proteopedia

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Revision as of 04:45, 28 April 2009

1 Overview
2 Structural Analysis
3 Functional Implications
- 3.1 Infectivity
- 3.2 Phage Display
4 Evolutionarily Related Proteins
- 4.1 N2
- 4.2 N1
5 Links to Available Structures
6 References

Overview

Figure Adapted from Hill and Stockley et al, 1996 ^[1]

Gene 3 protein (g3p pr pIII) is a minor coat protein found on the surface of filamentous bacteriophage ^[2]. The protein consists of 406 amino acids divided into three domains interspaced with glycine linkers ^[2] ^[3]. Peptides or proteins can be fused to g3p and evaluated for binding or other properties.

Structural Analysis

Five major papers will be discussed outlining the evolution of structure analysis of g3p.

Initial Observations

The first structure of g3p entered into the PDB was by Holliger and Riechmann in late 1996-early 1997^[4]. They used NMR spectroscopy to create a structure of the first domains of g3p. Shown is a combination of the 15 most energetically favorable states. Observations of secondary structure are below.

In 1998, Lubkowski et al crystallized the first two domains of g3p. They made two overall observations: (1) ^[2] and (2) ^[2] not found in other structures (Holliger et al)

D1 domain

The D1 domain consists of mostly beta sheets. Both Holliger and Riechmann as well as Lubowski et al noted a ^[2] in their respective publications^[4]^[2]. This aside, five arranged as a barrel-like motif, which participates with two other strands from second domain to make an antiparallel sheet ^[2]. Disulfide bonds exist between Cys 7 and Cys 36 (left handed helix) and Cys 46 and Cys 53 (right handed hook) ^[2].

N2 domain

- 8 beta strands, 6 in a mixed beta sheet, 2 interacting with N2 antiparallel sheet ^[2] Strand between B6 and B7 doesn’t have a specific motif but contributes via stabilizing hydrophobic interactions ^[2] 3 hairpins between 8 and 9, 9 and 10, and 10 and 11 (cis proline in the last hairpin) ^[2] One alpha helix that interacts with rest of N2 domain by hydrophobic interactions ^[2] Cation pi interaction between His 191 and Phe 199 C terminus of N2 has seven peptides, 3 of which are proline, 1 of which is in the cis conformation

Template:STRUCTURE 2g3p

Functional Implications

Infectivity

The linkers don’t have a specific purpose but appear to give the protein better flexibility providing optimal infectivity ^[2]. N1 interacts with TolA protein anchoring it to bacterial cell. ^[2]( and Cabilly)  see Riechmann and Holliger. The C terminal domain of TolA is the coreceptor for filamentous phage infection of E coli. Cell 90, 351-360 (1997) N1 and N2 have 15% identity ^[2] but a “nearly identical fold”, question of sharing common origins?? Possible gene duplication D2 domain binds to F pilus (Chatellier et al) D3 domain “anchors” to F pilus (Chatellier et al) and is necessary for phage packaging (Holliger et al) Infection with filamentous phage does not cause host cell lysis or death (Gailus and Rasched) Without N2 domain, infectivity is very low but initial contact is via N1 domain (Lubkowski et al). The complex formed by D1 and D2 may prevent destruction of the protein from bacterial proteases, Upon binding the protein opens up and D3 can then reach the inner membrane of the bacteria. (Chatellier). Horseshoe shaped molecule with polar molecules facing toward the center ^[2], this central area is thought to interact with pilus…however pilus is almost completely hydrophobic?? Peptides can be fused to CT domain or to the N1 domain, neither areas are near the central area of the horseshoe ^[2]. Amino terminus domain is necessary for infection, but full protein does not need to exist for all five particles on the surface (Cabilly). Fusions to N terminus (no affect on infectivity), between D12 and D3 (100 fold reduction for peptide insertion, and a 1000 to 100,000 fold for noncovalently interacting peptides)(Chatellier et al)

Phage Display

N terminus can be truncated and the peptide of choice can be inserted (Cabilly) Insertions can also be done between D2 and D3 (H and R, 9032075)

Evolutionarily Related Proteins

N1 and N2 have 15% identity ^[2] but a “nearly identical fold”, question of sharing common origins?? Possible gene duplication

Used DALI to identify similar proteins with both domains ^[2]  did not identify any When looking at each domain, they found some similar proteins

N2

PDZ domain of Human discs large protein (1pdr) Z score = 2.1) while smaller, 2 beta strands in core of domain share no identity with N2 ^[2](H and R, 9032075) PTB domain H and R, 9032075)

N1

Reported correlation with homopexin (1hxn) Z score 1.1 (p>0.05) ^[2] Compared with permuted SH3 domain (1tuc), but no sequence homology ^[2]

Links to Available Structures

References

↑ Hill HR, Stockley PG. Phage presentation. Mol Microbiol. 1996 May;20(4):685-92. PMID:8793867 doi:10.1111/j.1365-2958.1996.tb02508.x
↑ ^2.00 ^2.01 ^2.02 ^2.03 ^2.04 ^2.05 ^2.06 ^2.07 ^2.08 ^2.09 ^2.10 ^2.11 ^2.12 ^2.13 ^2.14 ^2.15 ^2.16 ^2.17 ^2.18 ^2.19 ^2.20 ^2.21 Lubkowski J, Hennecke F, Pluckthun A, Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p. Nat Struct Biol. 1998 Feb;5(2):140-7. PMID:9461080
↑ Cabilly S. The basic structure of filamentous phage and its use in the display of combinatorial peptide libraries. Mol Biotechnol. 1999 Sep;12(2):143-8. PMID:10596371 doi:10.1385/MB:12:2:143
↑ ^4.0 ^4.1 Holliger P, Riechmann L. A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd. Structure. 1997 Feb 15;5(2):265-75. PMID:9032075

Proteopedia Page Contributors and Editors (what is this?)

Leah Novinger, Michal Harel, Alexander Berchansky

Retrieved from "http://52.214.119.220/wiki/index.php/G3p"

@@ Line 11: / Line 11: @@
 ===Initial Observations===
 <applet load='1fgp' size='150' frame='true' align='left' caption='Initial structure of D1' />The first structure of g3p entered into the PDB was by Holliger and Riechmann in late 1996-early 1997<ref name="holliger 97"> PMID:9032075 </ref>.  They used NMR spectroscopy to create a structure of the first domains of g3p.  Shown is a combination of the 15 most energetically favorable states. Observations of secondary structure are below.
+<applet load='1g3p' size='300' frame='true' align='right' caption='Important characteristics of the N-terminal domains of g3p' />
 In 1998, Lubkowski et al crystallized the first two domains of g3p.  They made two overall observations:
-(1) <scene name='G3p/Pro_213_in_cis_conformation/1'>Cis proline near C terminal end</scene> <ref name="lubkowski"/> and (2) <scene name='G3p/Oxidized_tryptophan/1'>An oxidized tryptophan</scene> <ref name="lubkowski"/> not found in other structures (Holliger et al)<applet load='1g3p' size='300' frame='true' align='right' caption='Important characteristics of the N-terminal domains of g3p' />
+(1) <scene name='G3p/Pro_213_in_cis_conformation/1'>Cis proline near C terminal end</scene> <ref name="lubkowski"/> and (2) <scene name='G3p/Oxidized_tryptophan/1'>An oxidized tryptophan</scene> <ref name="lubkowski"/> not found in other structures (Holliger et al)
 ===D1 domain===
 The D1 domain consists of mostly beta sheets. Both Holliger and Riechmann as well as Lubowski et al noted a <scene name='G3p/N_terminal_alpha_helix/1'>N terminal alpha helix</scene> <ref name="lubkowski"/> in their respective publications<ref name="holliger 97"/><ref name="lubkowski"/>.  This aside, five <scene name='G3p/Beta_strands/1'>beta strands</scene> arranged as a barrel-like motif, which participates with two other strands from second domain to make an antiparallel sheet <ref name="lubkowski"/>. Disulfide bonds exist between Cys 7 and Cys 36 (left handed helix) and Cys 46 and Cys 53 (right handed hook) <ref name="lubkowski"/>.

G3p