G3p
From Proteopedia
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===Initial Observations=== | ===Initial Observations=== | ||
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. | 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. | ||
| - | |||
In 1997, Lubkowski et al crystallized the first two domains of g3p from M13 phage to 1.46Å resolution. Differences from the first paper published were mainly attributed to turns in the structure, the orientation of the C terminus, and interacting amino acids between the two domains. In addition, the authors made two overall observations: | In 1997, Lubkowski et al crystallized the first two domains of g3p from M13 phage to 1.46Å resolution. Differences from the first paper published were mainly attributed to turns in the structure, the orientation of the C terminus, and interacting amino acids between the two domains. In addition, the authors made two overall observations: | ||
(1) <scene name='G3p/Pro_213_in_cis_conformation/1'>Cis proline near C terminal end</scene> and (2) <scene name='G3p/Oxidized_tryptophan/1'>An oxidized tryptophan</scene> <ref name="lubkowski"/>. The cis proline identified will later turn out to be important for function of the protein. The oxidized tryptophan, however, was not found in other structures <ref name="holliger 99"> PMID: 10329170 </ref>. | (1) <scene name='G3p/Pro_213_in_cis_conformation/1'>Cis proline near C terminal end</scene> and (2) <scene name='G3p/Oxidized_tryptophan/1'>An oxidized tryptophan</scene> <ref name="lubkowski"/>. The cis proline identified will later turn out to be important for function of the protein. The oxidized tryptophan, however, was not found in other structures <ref name="holliger 99"> PMID: 10329170 </ref>. | ||
| - | One year later, another structure containing D1 and D2 (albeit from the filamentous phage, fd, containing a two residue difference) was published using x-ray crystallography to 1.9Å resolution <ref name="holliger 99"/>. Dimer formation was observed, but was attributed to the experimental conditions. | + | One year later, another structure containing D1 and D2 (albeit from the filamentous phage, fd, containing a two residue difference) was published using x-ray crystallography to 1.9Å resolution <ref name="holliger 99"/>. Dimer formation was observed, but was attributed to the experimental conditions. |
| + | |||
| + | Finally, two papers <ref name="lubkowski 99" PMID: 10404600 </ref><ref name="deprez" PMID:15701516</ref> were later published described interactions between g3p and TolA protein (located on the host cell, see ||Infectivity||. | ||
===D1 Domain=== | ===D1 Domain=== | ||
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===D3 Domain=== | ===D3 Domain=== | ||
| + | Upon review of the literature, no structural analyses of this domain were identified. | ||
===Linkers=== | ===Linkers=== | ||
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==Functional Implications== | ==Functional Implications== | ||
===Infectivity=== | ===Infectivity=== | ||
| + | {{STRUCTURE_1tol | PDB=1tol | SCENE= }} | ||
The function of the protein has a close correlation with its structural domains. The D1 domain interacts with TolA protein in the periplasm of the bacterial cell. <ref name="lubkowski"/><ref name="cabilly"/><ref name="holliger 99"/>. (The C terminal domain of TolA is the coreceptor for filamentous phage infection of E coli(Cell 90, 351-360 (1997)). The D2 domain binds to F pilus on the outer membrane of ''Escherichia coli'', however, it is also blocks TolA binding to D1 in the absence of the F pilus <ref name="chatellier"> PMID:10606756 </ref>. In fact, without the D2 domain, infectivity is very low<ref name="lubkowski"/>. It has been speculated that D2 interacts with the F pilus first, drawing the phage closer to the bacterial cell, thus allowing D1-TolA interactions to occur<ref name="holliger 99"/>. Chatellier et al suggest that the complex formed by D1 and D2 may prevent destruction of the protein from bacterial proteases, and upon binding the protein opens up and D3 can then reach the inner membrane of the bacteria<ref name="chatellier"/>. Lubkowski et al like the interaction between D1 and D2 to a horseshoe shaped molecule attributing <scene name='G3p/Hydrophobic/1'>hydrophobic molecules </scene> facing toward the center as stabilizing factors <ref name="lubkowski"/>. | The function of the protein has a close correlation with its structural domains. The D1 domain interacts with TolA protein in the periplasm of the bacterial cell. <ref name="lubkowski"/><ref name="cabilly"/><ref name="holliger 99"/>. (The C terminal domain of TolA is the coreceptor for filamentous phage infection of E coli(Cell 90, 351-360 (1997)). The D2 domain binds to F pilus on the outer membrane of ''Escherichia coli'', however, it is also blocks TolA binding to D1 in the absence of the F pilus <ref name="chatellier"> PMID:10606756 </ref>. In fact, without the D2 domain, infectivity is very low<ref name="lubkowski"/>. It has been speculated that D2 interacts with the F pilus first, drawing the phage closer to the bacterial cell, thus allowing D1-TolA interactions to occur<ref name="holliger 99"/>. Chatellier et al suggest that the complex formed by D1 and D2 may prevent destruction of the protein from bacterial proteases, and upon binding the protein opens up and D3 can then reach the inner membrane of the bacteria<ref name="chatellier"/>. Lubkowski et al like the interaction between D1 and D2 to a horseshoe shaped molecule attributing <scene name='G3p/Hydrophobic/1'>hydrophobic molecules </scene> facing toward the center as stabilizing factors <ref name="lubkowski"/>. | ||
| - | {{STRUCTURE_1tol | PDB=1tol | SCENE= }} | ||
| - | The function of D3 was elicited last. 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). | ||
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| + | The function of D3 was elicited last. D3 domain “anchors” to F pilus and is necessary for phage packaging<ref name="holliger 99"/>. | ||
===Phage Display=== | ===Phage Display=== | ||
| + | Infection with filamentous phage does not cause host cell lysis or death (Gailus and Rasched). | ||
N terminus can be truncated and the peptide of choice can be inserted (Cabilly) | 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) | Insertions can also be done between D2 and D3 (H and R, 9032075) | ||
Revision as of 16:45, 28 April 2009
Contents |
Overview
Gene 3 protein (g3p pr pIII) is a minor coat protein found on the surface of filamentous bacteriophage [1]. The protein consists of 406 amino acids divided into three domains interspaced with glycine linkers [1] [2]. 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 [3].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 1997, Lubkowski et al crystallized the first two domains of g3p from M13 phage to 1.46Å resolution. Differences from the first paper published were mainly attributed to turns in the structure, the orientation of the C terminus, and interacting amino acids between the two domains. In addition, the authors made two overall observations: (1) and (2) [1]. The cis proline identified will later turn out to be important for function of the protein. The oxidized tryptophan, however, was not found in other structures [4].
One year later, another structure containing D1 and D2 (albeit from the filamentous phage, fd, containing a two residue difference) was published using x-ray crystallography to 1.9Å resolution [4]. Dimer formation was observed, but was attributed to the experimental conditions.
Finally, two papers Cite error: Invalid <ref> tag;
invalid names, e.g. too many were later published described interactions between g3p and TolA protein (located on the host cell, see ||Infectivity||.
D1 Domain
The first structure of g3p entered into the PDB was by Holliger and Riechmann in late 1996-early 1997[3]. The consists of mostly beta sheets. Both Holliger and Riechmann as well as Lubkowski et al noted a in their respective publications[3][1]. This aside, five arranged as a barrel-like motif, which participates with two other strands from second domain to make an antiparallel sheet. Disulfide bonds exist between Cys 7 and Cys 36 (left handed helix) and Cys 46 and Cys 53 (right handed hook) [1].
D2 Domain
Template:STRUCTURE 2g3p This domain contains eight beta strands: six in a mixed beta sheet and two interacting with D1 antiparallel sheet (β6 and β13 [1]. The amino acids between β6 and β7 doesn’t have a specific motif but has stabilizing hydrophobic interactions with other parts of the domain [1]. Three hairpins exist in this domain: between β8 and β9, β9 and β10, and β10 and β11 (cis proline in the last hairpin) [1]. The final secondary structural element is an alpha helix that interacts with rest of the domain via hydrophobic interactions [1]. Of note, there is a cation-π interaction between His 191 and Phe 199. The C terminus of D2 has seven peptides, 3 of which are proline, 1 of which is [1].
Further elaboration on the interactions between the domains is described in #Infectivity
D3 Domain
Upon review of the literature, no structural analyses of this domain were identified.
Linkers
The linkers don’t have a specific purpose but appear to give the protein better flexibility providing optimal infectivity [1]. In both Lubkowski et al and Holliger et al, these regions were not observed through crystallography study [1][4].
Functional Implications
Infectivity
Template:STRUCTURE 1tol The function of the protein has a close correlation with its structural domains. The D1 domain interacts with TolA protein in the periplasm of the bacterial cell. [1][2][4]. (The C terminal domain of TolA is the coreceptor for filamentous phage infection of E coli(Cell 90, 351-360 (1997)). The D2 domain binds to F pilus on the outer membrane of Escherichia coli, however, it is also blocks TolA binding to D1 in the absence of the F pilus [5]. In fact, without the D2 domain, infectivity is very low[1]. It has been speculated that D2 interacts with the F pilus first, drawing the phage closer to the bacterial cell, thus allowing D1-TolA interactions to occur[4]. Chatellier et al suggest that the complex formed by D1 and D2 may prevent destruction of the protein from bacterial proteases, and upon binding the protein opens up and D3 can then reach the inner membrane of the bacteria[5]. Lubkowski et al like the interaction between D1 and D2 to a horseshoe shaped molecule attributing facing toward the center as stabilizing factors [1].
The function of D3 was elicited last. D3 domain “anchors” to F pilus and is necessary for phage packaging[4].
Phage Display
Infection with filamentous phage does not cause host cell lysis or death (Gailus and Rasched). 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) Peptides can be fused to CT domain or to the N1 domain, neither areas are near the central area of the horseshoe [1].
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)
Evolutionarily Related Proteins
N1 and N2 have 15% identity but a “nearly identical fold”, question of sharing common origins or a gene duplication[1].
Used DALI to identify similar proteins with both domains, Lubkowski et al were unable to identify any evolutionarily related proteins[1]. When looking at individual domains, they found some similar proteins.
D1
They reported a correlation with homopexin [(1hxn)] Z score 1.1 (p>0.05)[1]. A comparison with a permuted SH3 domain [(1tuc)] was made, but sequence homology did not exist[1].
D2
Lubkowski et al identified the PDZ domain of Human discs large protein (1pdr) as a potentially related protein (Z score = 2.1). This protein is smaller than g3p, and consequently two beta strands in th core of domain share no identity with D2 [1].(H and R, 9032075)
PTB domain H and R, 9032075)
Links to Available Structures
PDB [1fgp] [1g3p] [2g3p] [1tol] [1s62]
References
- ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 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
- ↑ 2.0 2.1 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
- ↑ 3.0 3.1 3.2 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
- ↑ 4.0 4.1 4.2 4.3 4.4 4.5 Holliger P, Riechmann L, Williams RL. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability. J Mol Biol. 1999 May 14;288(4):649-57. PMID:10329170 doi:10.1006/jmbi.1999.2720
- ↑ 5.0 5.1 Chatellier J, Hartley O, Griffiths AD, Fersht AR, Winter G, Riechmann L. Interdomain interactions within the gene 3 protein of filamentous phage. FEBS Lett. 1999 Dec 17;463(3):371-4. PMID:10606756
