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4Q7Q Structure and Proposed Functionality

(NOTE TO ALL EDITORS: This page is part of a final project for a biochemistry lab at Elizabethtown College. Please do not edit this.)

4Q7Q is a homodimeric protein complex that originates from the bacterial species Chitinophaga Pinensis and has a mass of 58.5 kDa. It is a member of the SGNH Hydrolase Superfamily with structural and sequential similarities to esterases and lipases. Current evidence suggests it causes the hydrolysis of esters and/or acetyl groups on lipids/lipid-like molecules via a serine protease-like active site.

1 Overview
2 Functionality Overview
3 Sequence Analysis
4 Structural Analysis
5 Substrates
6 Experimental Data

Overview

General Structure and Origins

4Q7Q exists as a homodimer Quaternary structure.^[1] Analyzing primary and Quaternary structures of 4Q7Q with SPRITE and Chimera revealed two chains identical in both shape and sequence^[2] Each chain is 266 residues long, and the entire complex has a molecular weight of approximately 58.5 kDa.^[1].

Primary Sequences of the A and B chains of 4Q7Q.^[2].

4Q7Q proteins primarily appear in bacterial species.^[3]. Research reveals how nearly every enzyme with similar sequencing to 4Q7Q is found in bacterium, with a slight exception to eukaryotes.^[4] Additionally, the PDB entry for 4Q7Q notes how it potentially can be found in Chitinophaga pinensis, a gram-negative bacterial species which can degrade chitin.^[3].^[5].

Family and Superfamily

Research shows that 4Q7Q is a member of the SGNH Hydrolase protein super family. BLAST and InterPro research suggested 4Q7Q best fits this superfamily, and the known conserved residues seen from SPRITE analysis—Serine, Glycine, Asparagine, and Histidine—line up with those observed throughout this family.^[4]^[6]. Notably, this superfamily is also referred to as the GDSL Hydrolase superfamily.D,E^[4]^[6].

Regarding what protein family 4Q7Q belongs to, DALI results suggest it is a part of a sub-family of the greater GDSL/SGNH superfamily. A PDB90% DALI search labels 4Q7Q as a part of the “Lipolytic Protein G-D-S-L Family,” which refers to enzymes that hydrolyze lipid substrates.^[7].

Structure/Sequence Analysis

Functionality Overview

We currently believe 4Q7Q hydrolyzes fatty acid-like structures

Sequence Analysis

The primary sequence of 4Q7Q shows several conserved sequences between it and esterase-like proteins. A sequence of GDSI—similar to the GDSL sequence seen from its family and superfamily—can be seen between 4Q7Q and enzymes like Isoamyl Acetate-Hydrolyzing Esterase. Other noteworthy conserved sequences between esterases and 4Q7Q include GxND and DGxH.

These enzymes also share similar secondary structures. Segments of alpha-helixes and beta-sheet strands appear and remain nearly entirely conserved throughout esterase analysis. A few conserved coils appear, but these sections do not appear as often as the other two secondary structures.

Similar conserved sequences could be found between 4Q7Q and lipases. The GDSI, GxND, and DGxH sequences can be seen from lipases like 7BXD.? The same secondary structure segments can also be located in the lipases analyzed.

Structural Analysis

Right-hand SPRITE analysis revealed 4Q7Q exhibited residues like those seen from enzymes operating with acetyl-like substrates. Specifically, residues Ser. 30, Gly. 69, Asn. 97, Asp. 251, and His. 254 on the A and B chains of 4Q7Q line up with similarly positioned residues on esterases like Platelet-Activating Factor Acetylhyd (PAFA), which exhibited an RMSD of 0.25 angstroms when compared to 4Q7Q.

Other proteins with similar motifs of note are Thioesterase I and Rhamnogalacturonan Acetylesterase, with RMSD values of 0.46 and 0.61, respectively. These alignments focus on the same active site as PAFA did, suggesting the acetyl-like substrates 4Q7Q focuses on are similar to esters.

PFAM graphics from DALI revealed significant structural equivalence between 4Q7Q, a lipase-like protein, Rhamnogalacturonan Acetylesterase, and Sialate O-acetylesterase.

4Q7Q’s inclusion in this family also supports its SPRITE-derived hypothetical functionality. Rhamnogalacturonan Acetylesterase—the enzyme with one of the best SPRITE-based alignment relative to 4Q7Q—is a member of this family.F Proteins in this family are also known for containing a “unique hydrogen bond network that [stabilizes]” the active site.F

Substrates

SwissDock analysis showed a preference for larger molecules, specifically fatty acids. Lactide, Ethyl Butyrate, and Triethylene Glycol exhibited noticeably weak binding affinities to the theorized active site of 4Q7Q. These ligands may be ill-suited to act as substrates for 4Q7Q as they are remarkably polar, and lipids—one of the potential categories of substrates for 4Q7Q—are mostly non-polar.

Despite this, these ligands show noticeable hydrophobic interactions with the active site. This implies 4Q7Q uses hydrophobic regions to help guide substrates into the right orientation for enzymatic processes. This also further supports the possibility that 4Q7Q primarily operates with hydrophobic lipid-based substrates. This also explains why Methyl Acetate exhibited a relatively weaker affinity for 4Q7Q, as its smaller structure prevented hydrophobic interactions.

Experimental Data

References

↑ ^1.0 ^1.1 4Q7Q. Protein Database, 2014. https://www.rcsb.org/structure/4Q7Q
↑ ^2.0 ^2.1 Nadzirin, N.; Gardiner, E.; Willett, P.; Artymiuk, P. J.; Firdaus-Raih, M. 2012. SPRITE and ASSAM: web servers for side chain 3D-motif searching in protein structures. Nucleic Acids Res., 40(Web Server Issue), W380-6.
↑ ^3.0 ^3.1 Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/
↑ ^4.0 ^4.1 ^4.2 SGNH hydrolase superfamily. InterPro, 2017. https://www.ebi.ac.uk/interpro/entry/InterPro/IPR036514/
↑ Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/
↑ ^6.0 ^6.1 Molgaard, A.; Kauppinen, S.; Larsen, S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Struct., 2000, 8(4), 373-383. https://www.sciencedirect.com/science/article/pii/S0969212600001180?via%3Dihub
↑ Akoh, C. C.; Lee, G.; Liaw, Y.; Huang, T.; Shaw, J. GDSL family of serine esterases/lipases. Prog. Lipid Res., 2004, 43(6), 534-552. https://pubmed.ncbi.nlm.nih.gov/15522763/

1. 4Q7Q. Protein Database, 2014. 2. Nadzirin, N.; Gardiner, E.; Willett, P.; Artymiuk, P. J.; Firdaus-Raih, M. 2012. SPRITE and ASSAM: web servers for side chain 3D-motif searching in protein structures. Nucleic Acids Res., 40(Web Server Issue), W380-6.

External Resources

1. https://www.rcsb.org/structure/4Q7Q

A) 1WAB. Protein Database, 1997. https://www.rcsb.org/structure/1WAB B) Ho, Y. S.; Sewnson, L.; Derewenda, U.; Serre, L.; Wei, Y.; Dauter, Z.; Hattori, M.; Adachi, T.; Aoki, J.; Arai, H.; Inoue, K.; Derewenda, Z. S. Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer. Nature, 1997, 385, 89-93. https://www.nature.com/articles/385089a0 https://www.nature.com/articles/385089a0 C) Miesfeld, R. L.; McEvoy, M. M. Biochemistry, 2nd ed.; W. W. Norton & Company, 2021. D) SGNH hydrolase superfamily. InterPro, 2017. https://www.ebi.ac.uk/interpro/entry/InterPro/IPR036514/ E) Molgaard, A.; Kauppinen, S.; Larsen, S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Struct., 2000, 8(4), 373-383. https://www.sciencedirect.com/science/article/pii/S0969212600001180?via%3Dihub F) 4Q7Q. Protein Database, 2014. https://www.rcsb.org/structure/4Q7Q G) Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/ H) Akoh, C. C.; Lee, G.; Liaw, Y.; Huang, T.; Shaw, J. GDSL family of serine esterases/lipases. Prog. Lipid Res., 2004, 43(6), 534-552. https://pubmed.ncbi.nlm.nih.gov/15522763/ I) 7BXD. Protein Database, 2021. https://www.rcsb.org/structure/7BXD J) Madej,T.; Lanczycki, C. J.; Zhang, D.; Thiessen, P. A.; Geer, R. C.; Marchler-Bauer, A.; Bryant, S. H. MMDB and VAST+: tracking structural similarities between macromolecular complexes. Nucleic Acids Res., 2014, 42(Database), D297-303. https://doi.org/10.1093/nar/gkt1208

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@@ Line 15: / Line 15: @@
 [[Image:4Q7QABSequence.png|400px|left|thumb| Primary Sequences of the A and B chains of 4Q7Q.<ref name="SPRITE" />.]]
-Q7Q proteins primarily appear in bacterial species.<ref name="Origin">Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/</ref>. Research reveals how nearly every enzyme with similar sequencing to 4Q7Q is found in bacterium, with a slight exception to eukaryotes.<ref name="SGNH">SGNH hydrolase superfamily. InterPro, 2017. https://www.ebi.ac.uk/interpro/entry/InterPro/IPR036514/</ref> Additionally, the PDB entry for 4Q7Q notes how it potentially can be found in Chitinophaga pinensis, a gram-negative bacterial species which can degrade chitin.<ref name="Origin" />. <ref name="GDSL">Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/ </ref>.
+Q7Q proteins primarily appear in bacterial species.<ref name="Origin">Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/</ref>. Research reveals how nearly every enzyme with similar sequencing to 4Q7Q is found in bacterium, with a slight exception to eukaryotes.<ref name="SGNH">SGNH hydrolase superfamily. InterPro, 2017. https://www.ebi.ac.uk/interpro/entry/InterPro/IPR036514/</ref> Additionally, the PDB entry for 4Q7Q notes how it potentially can be found in Chitinophaga pinensis, a gram-negative bacterial species which can degrade chitin.<ref name="Origin" />.<ref name="GDSL">Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/ </ref>.
 === Family and Superfamily ===
-Research shows that 4Q7Q is a member of the SGNH Hydrolase protein super family. BLAST and InterPro both suggested 4Q7Q’s inclusion in this family, and the known conserved residues seen from SPRITE analysis—Serine, Glycine, Asparagine, and Histidine—line up with those observed throughout this family.D,E Notably, this superfamily is also referred to as the GDSL Hydrolase superfamily.D,E
+Research shows that 4Q7Q is a member of the SGNH Hydrolase protein super family. BLAST and InterPro research suggested 4Q7Q best fits this superfamily, and the known conserved residues seen from SPRITE analysis—Serine, Glycine, Asparagine, and Histidine—line up with those observed throughout this family.<ref name="SGNH" /><ref name = "Molgaard">Molgaard, A.; Kauppinen, S.; Larsen, S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Struct., 2000, 8(4), 373-383. https://www.sciencedirect.com/science/article/pii/S0969212600001180?via%3Dihub</ref>.  Notably, this superfamily is also referred to as the GDSL Hydrolase superfamily.D,E<ref name="SGNH" /><ref name="Molgaard" />.
-Q7Q’s inclusion in this family also supports its SPRITE-derived hypothetical functionality. Rhamnogalacturonan Acetylesterase—the enzyme with one of the best SPRITE-based alignment relative to 4Q7Q—is a member of this family.F Proteins in this family are also known for containing a “unique hydrogen bond network that [stabilizes]” the active site.F
+Regarding what protein family 4Q7Q belongs to, DALI results suggest it is a part of a sub-family of the greater GDSL/SGNH superfamily. A PDB90% DALI search labels 4Q7Q as a part of the “Lipolytic Protein G-D-S-L Family,” which refers to enzymes that hydrolyze lipid substrates.<ref name="Akoh">Akoh, C. C.; Lee, G.; Liaw, Y.; Huang, T.; Shaw, J. GDSL family of serine esterases/lipases. Prog. Lipid Res., 2004, 43(6), 534-552. https://pubmed.ncbi.nlm.nih.gov/15522763/</ref>.
+=== Structure/Sequence Analysis ===
+== Functionality Overview ==
-Regarding what protein family 4Q7Q belongs to, DALI results suggest it is a part of a sub-family of the greater GDSL/SGNH superfamily. A PDB90% DALI search labels 4Q7Q as a part of the “Lipolytic Protein G-D-S-L Family,” which refers to enzymes that hydrolyze lipid substrates.I
+We currently believe 4Q7Q hydrolyzes fatty acid-like structures
 == Sequence Analysis ==
@@ Line 40: / Line 44: @@
 PFAM graphics from DALI revealed significant structural equivalence between 4Q7Q, a lipase-like protein, Rhamnogalacturonan Acetylesterase, and Sialate O-acetylesterase.
+Q7Q’s inclusion in this family also supports its SPRITE-derived hypothetical functionality. Rhamnogalacturonan Acetylesterase—the enzyme with one of the best SPRITE-based alignment relative to 4Q7Q—is a member of this family.F Proteins in this family are also known for containing a “unique hydrogen bond network that [stabilizes]” the active site.F
 == Substrates ==
@@ Line 47: / Line 53: @@
 Despite this, these ligands show noticeable hydrophobic interactions with the active site. This implies 4Q7Q uses hydrophobic regions to help guide substrates into the right orientation for enzymatic processes. This also further supports the possibility that 4Q7Q primarily operates with hydrophobic lipid-based substrates. This also explains why Methyl Acetate exhibited a relatively weaker affinity for 4Q7Q, as its smaller structure prevented hydrophobic interactions.
-== Theoretical Functionality and Proposed Bodily Purpose ==
+== Experimental Data ==
-Highlight the data that helped you come to your conclusion here including any relevant figures. Make sure include potential substrates and binding sites.