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A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

Lee Y, Mick J, Furdui C, Beamer LJ - PLoS ONE (2012)

Bottom Line: For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme.The results of these studies are interpreted in the context of structural and functional data on PMM/PGM.Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, Columbia, Missouri, United States of America.

ABSTRACT
Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

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Structural context of the coevolutionary residue network.(A) Ribbon diagram of PMM/PGM from P. aeruginosa (PDB ID 1K2Y) with a semi-transparent surface. Domain 4 (residues 369–463) of the protein is shown in pink; the first three domains are gray. The 12 top clique residues identified by the coevolutionary analysis are highlighted as space filling models in blue (domains 1–3) and magenta (domain 4). (B) Same as panel A, but rotated by 90° for an alternate view of the domain 4 interface. Note that the top clique residues in the interface form a contiguous patch that spans the width of the interface. (C) Ribbon diagram of P. aeruginosa PMM/PGM (PDB ID 1P5G) colored according to sequence conservation. Glucose 1-phosphate is shown as a stick model with green carbons. Conservation is calculated according to an entropy score (see Methods and Fig. 1B) where blue color indicates high and red indicates low conservation. Residues that were more than 10% gapped were assigned a value of 1. Figure made with PYMOL [63].
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pone-0038114-g003: Structural context of the coevolutionary residue network.(A) Ribbon diagram of PMM/PGM from P. aeruginosa (PDB ID 1K2Y) with a semi-transparent surface. Domain 4 (residues 369–463) of the protein is shown in pink; the first three domains are gray. The 12 top clique residues identified by the coevolutionary analysis are highlighted as space filling models in blue (domains 1–3) and magenta (domain 4). (B) Same as panel A, but rotated by 90° for an alternate view of the domain 4 interface. Note that the top clique residues in the interface form a contiguous patch that spans the width of the interface. (C) Ribbon diagram of P. aeruginosa PMM/PGM (PDB ID 1P5G) colored according to sequence conservation. Glucose 1-phosphate is shown as a stick model with green carbons. Conservation is calculated according to an entropy score (see Methods and Fig. 1B) where blue color indicates high and red indicates low conservation. Residues that were more than 10% gapped were assigned a value of 1. Figure made with PYMOL [63].

Mentions: The locations of the 12 top clique residues on the structure of P. aeruginosa PMM/PGM are shown in Fig. 3A. With only a few exceptions, it can be clearly seen that the top clique residues localize to a small region of the structure: the interface between domain 4 (pink) and the rest of the protein. The top clique residues fall on both sides of this interface, including residues from domains 3 (261, 284, 285) and 4 (374, 410, 419, 430, 432) of the protein. Indeed, these eight residues form an essentially contiguous residue patch (Fig. 2B), which spans the width (short dimension) of the domain interface (Fig. 3B). The remaining residues (those outside the domain 4 interface) are found in domains 1 (88), 2 (249), and elsewhere in domain 3 (331, 341), generally near the center of the molecule, but not within the active site cleft. With the exception of K285, which makes ligand contacts in certain enzyme-substrate complexes [12], none of the top clique residues were of previously implicated functional significance in PMM/PGM. However, the domain 4 interface is a known site of conformational change of the protein, as first observed in crystallographic studies of enzyme-substrate complexes (see Fig. S1 and Discussion for more detail) [12].


A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

Lee Y, Mick J, Furdui C, Beamer LJ - PLoS ONE (2012)

Structural context of the coevolutionary residue network.(A) Ribbon diagram of PMM/PGM from P. aeruginosa (PDB ID 1K2Y) with a semi-transparent surface. Domain 4 (residues 369–463) of the protein is shown in pink; the first three domains are gray. The 12 top clique residues identified by the coevolutionary analysis are highlighted as space filling models in blue (domains 1–3) and magenta (domain 4). (B) Same as panel A, but rotated by 90° for an alternate view of the domain 4 interface. Note that the top clique residues in the interface form a contiguous patch that spans the width of the interface. (C) Ribbon diagram of P. aeruginosa PMM/PGM (PDB ID 1P5G) colored according to sequence conservation. Glucose 1-phosphate is shown as a stick model with green carbons. Conservation is calculated according to an entropy score (see Methods and Fig. 1B) where blue color indicates high and red indicates low conservation. Residues that were more than 10% gapped were assigned a value of 1. Figure made with PYMOL [63].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369874&req=5

pone-0038114-g003: Structural context of the coevolutionary residue network.(A) Ribbon diagram of PMM/PGM from P. aeruginosa (PDB ID 1K2Y) with a semi-transparent surface. Domain 4 (residues 369–463) of the protein is shown in pink; the first three domains are gray. The 12 top clique residues identified by the coevolutionary analysis are highlighted as space filling models in blue (domains 1–3) and magenta (domain 4). (B) Same as panel A, but rotated by 90° for an alternate view of the domain 4 interface. Note that the top clique residues in the interface form a contiguous patch that spans the width of the interface. (C) Ribbon diagram of P. aeruginosa PMM/PGM (PDB ID 1P5G) colored according to sequence conservation. Glucose 1-phosphate is shown as a stick model with green carbons. Conservation is calculated according to an entropy score (see Methods and Fig. 1B) where blue color indicates high and red indicates low conservation. Residues that were more than 10% gapped were assigned a value of 1. Figure made with PYMOL [63].
Mentions: The locations of the 12 top clique residues on the structure of P. aeruginosa PMM/PGM are shown in Fig. 3A. With only a few exceptions, it can be clearly seen that the top clique residues localize to a small region of the structure: the interface between domain 4 (pink) and the rest of the protein. The top clique residues fall on both sides of this interface, including residues from domains 3 (261, 284, 285) and 4 (374, 410, 419, 430, 432) of the protein. Indeed, these eight residues form an essentially contiguous residue patch (Fig. 2B), which spans the width (short dimension) of the domain interface (Fig. 3B). The remaining residues (those outside the domain 4 interface) are found in domains 1 (88), 2 (249), and elsewhere in domain 3 (331, 341), generally near the center of the molecule, but not within the active site cleft. With the exception of K285, which makes ligand contacts in certain enzyme-substrate complexes [12], none of the top clique residues were of previously implicated functional significance in PMM/PGM. However, the domain 4 interface is a known site of conformational change of the protein, as first observed in crystallographic studies of enzyme-substrate complexes (see Fig. S1 and Discussion for more detail) [12].

Bottom Line: For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme.The results of these studies are interpreted in the context of structural and functional data on PMM/PGM.Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, Columbia, Missouri, United States of America.

ABSTRACT
Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

Show MeSH
Related in: MedlinePlus