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Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase.

Langkilde A, Kristensen SM, Lo Leggio L, Mølgaard A, Jensen JH, Houk AR, Navarro Poulsen JC, Kauppinen S, Larsen S - Acta Crystallogr. D Biol. Crystallogr. (2008)

Bottom Line: The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop.The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures.Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.

ABSTRACT
An extremely low-field signal (at approximately 18 p.p.m.) in the (1)H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental (1)H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 A resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK(a) values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

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Examples of different types of short hydrogen bonds from the RGAE D192N structure. (a) Asp75–Asp87, (b) Thr10–Asp8, (c) Thr20–Gly17, (d) Val3–Thr34, (e) His169–Glu70 and Ser131–Glu70, (f) Arg46–Asp82 and Ser98–Asp82.
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fig6: Examples of different types of short hydrogen bonds from the RGAE D192N structure. (a) Asp75–Asp87, (b) Thr10–Asp8, (c) Thr20–Gly17, (d) Val3–Thr34, (e) His169–Glu70 and Ser131–Glu70, (f) Arg46–Asp82 and Ser98–Asp82.

Mentions: O—H⋯O hydrogen bonds with O⋯O distances shorter than 2.75 Å represent three different types of interactions. In accordance with the results from the analysis of small-molecule structures, the shortest is between the carboxy groups of Asp75 and Asp87 (Fig. 6 ▶ a), 2.47 (1) and 2.48 (2) Å in wild-type RGAE and RGAE D192N, respectively. The similar interaction between Glu202 and Glu206 observed in the orthorhombic structure of the wild type is not observed in the RGAE D192N structure, where the residues are involved in crystal contacts as described in §3.1.1. The two other types of short hydrogen bonds have side-chain OH groups as donor. Five hydrogen bonds with O⋯O distances in the range 2.56 (2)–2.75 (1) Å connect Ser/Thr side chains with the carboxylate groups from Asp/Glu residues in both RGAE structures. The O⋯O distances in the two structures are virtually identical, with one distance almost as short as the O⋯O distance in the carboxylic acid carboxylate hydrogen bond and significantly below the average distance of the similar interaction in small-molecule structures at 2.74 (10) Å. Two of these interactions are completely buried in the protein [Thr10–Asp8 (Fig. 6 ▶ b) and Ser131–Glu70]. Although the O⋯O distances could suggest that the carboxylic acid group functions as the hydrogen-bond donor, the predicted pK a values show unambigously that the OH groups are the proton donors, illustrating the significance of combining different information in the analysis of hydrogen bonds in proteins. The third group comprises hydrogen-bond interactions between OH groups and backbone amide O atoms. The interaction between Tyr30 and Glu202 is only observed in wild-type RGAE. In RGAE D192N Glu202 is involved in crystal packing, which explains why this hydrogen bond is not formed. The O⋯O distances vary between 2.62 (3) and 2.87 (2) Å, with the equivalent distances in the two structures being remarkably similar. In the small-molecule structures the average O⋯O distance is 2.77 (8) Å, with a fairly large spread (Fig. 5 ▶). The abundance and short distances made us look for a possible structural role for these inter­actions. Four interactions of this type connect residues in loops. The hydrogen bonds Thr86–Gly76, Ser44–Arg85 and Ser44–Arg85 connect different loops and Thr20–Gly17 (Fig. 6 ▶ c) forms a link between residues in the same loop. The three other hydrogen bonds between residues close in the sequence (Thr215–Ala211, Ser187–Thr184 and Thr49–Ala45) connect residues in the same α-­helix and may have a role in reducing the solvent-exposure of the helix.


Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase.

Langkilde A, Kristensen SM, Lo Leggio L, Mølgaard A, Jensen JH, Houk AR, Navarro Poulsen JC, Kauppinen S, Larsen S - Acta Crystallogr. D Biol. Crystallogr. (2008)

Examples of different types of short hydrogen bonds from the RGAE D192N structure. (a) Asp75–Asp87, (b) Thr10–Asp8, (c) Thr20–Gly17, (d) Val3–Thr34, (e) His169–Glu70 and Ser131–Glu70, (f) Arg46–Asp82 and Ser98–Asp82.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Examples of different types of short hydrogen bonds from the RGAE D192N structure. (a) Asp75–Asp87, (b) Thr10–Asp8, (c) Thr20–Gly17, (d) Val3–Thr34, (e) His169–Glu70 and Ser131–Glu70, (f) Arg46–Asp82 and Ser98–Asp82.
Mentions: O—H⋯O hydrogen bonds with O⋯O distances shorter than 2.75 Å represent three different types of interactions. In accordance with the results from the analysis of small-molecule structures, the shortest is between the carboxy groups of Asp75 and Asp87 (Fig. 6 ▶ a), 2.47 (1) and 2.48 (2) Å in wild-type RGAE and RGAE D192N, respectively. The similar interaction between Glu202 and Glu206 observed in the orthorhombic structure of the wild type is not observed in the RGAE D192N structure, where the residues are involved in crystal contacts as described in §3.1.1. The two other types of short hydrogen bonds have side-chain OH groups as donor. Five hydrogen bonds with O⋯O distances in the range 2.56 (2)–2.75 (1) Å connect Ser/Thr side chains with the carboxylate groups from Asp/Glu residues in both RGAE structures. The O⋯O distances in the two structures are virtually identical, with one distance almost as short as the O⋯O distance in the carboxylic acid carboxylate hydrogen bond and significantly below the average distance of the similar interaction in small-molecule structures at 2.74 (10) Å. Two of these interactions are completely buried in the protein [Thr10–Asp8 (Fig. 6 ▶ b) and Ser131–Glu70]. Although the O⋯O distances could suggest that the carboxylic acid group functions as the hydrogen-bond donor, the predicted pK a values show unambigously that the OH groups are the proton donors, illustrating the significance of combining different information in the analysis of hydrogen bonds in proteins. The third group comprises hydrogen-bond interactions between OH groups and backbone amide O atoms. The interaction between Tyr30 and Glu202 is only observed in wild-type RGAE. In RGAE D192N Glu202 is involved in crystal packing, which explains why this hydrogen bond is not formed. The O⋯O distances vary between 2.62 (3) and 2.87 (2) Å, with the equivalent distances in the two structures being remarkably similar. In the small-molecule structures the average O⋯O distance is 2.77 (8) Å, with a fairly large spread (Fig. 5 ▶). The abundance and short distances made us look for a possible structural role for these inter­actions. Four interactions of this type connect residues in loops. The hydrogen bonds Thr86–Gly76, Ser44–Arg85 and Ser44–Arg85 connect different loops and Thr20–Gly17 (Fig. 6 ▶ c) forms a link between residues in the same loop. The three other hydrogen bonds between residues close in the sequence (Thr215–Ala211, Ser187–Thr184 and Thr49–Ala45) connect residues in the same α-­helix and may have a role in reducing the solvent-exposure of the helix.

Bottom Line: The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop.The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures.Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.

ABSTRACT
An extremely low-field signal (at approximately 18 p.p.m.) in the (1)H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental (1)H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 A resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK(a) values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

Show MeSH
Related in: MedlinePlus