On the Importance of Carbohydrate-Aromatic Interactions for the Molecular Recognition of Chitooligosaccharides by Hevein Domains. NMR Studies of the Structure and Binding Affinity of AcAMP2-Like Peptides with non Natural Napthyl and Fluoroaromatic Residues

Residue Phe18 have been mutated to beta-(2-naphthyl)-alanine in the molecule studied here.

The atom HG2 of GLN17 is upfield shifted (at 0.459); residue 17 is shielded due the proximity to the naphtyl aromatic system of the residue 18.

Information obtained from PDB's Chemical Component Dictionary at http://wwpdb-remediation.rutgers.edu/downloads.html Downloaded on Mon Aug 8 12:36:19 2011

Information obtained from PDB's Chemical Component Dictionary at http://wwpdb-remediation.rutgers.edu/downloads.html Downloaded on Mon Aug 8 12:37:48 2011

(2S)-2-amino-3-(naphthalen-2-yl)propanoic acid (non-preferred name)

(2S)-2-azanyl-3-naphthalen-2-yl-propanoic acid

Chemistry (Weinheim an der Bergstrasse, Germany)
On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues.

The conformation in water of antimicrobial protein 2 from Amaranthus caudatus (Ac-AMP2) was determined using 1H NMR, DIANA and restrained molecular modeling. Ac-AMP2 is a 30 amino acid residue, lectin-like protein that specifically binds to chitin, a polymer of beta-1,4-N-acetyl-D-glucosamine. After sequence specific resonance assignments, a total of 198 distance restraints were collected from 2D NOESY buildup spectra at 500 MHz at pH 2, supplemented by a 2D NOESY spectrum at 600 MHz. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of Ac-AMP2 is presented as a set of 26 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol in the AMBER force field, with a backbone r.m.s.d. for the well defined Glu3-Cys28 segment of 0.69(+/-0.12) angstroms. The main structural element is an antiparallel beta-sheet from Met13 to Lys23 including a betaI-turn over Gln17-Phel8 with a beta bulge at Gly19. In addition, a beta'I turn over Arg6-Gly7, a beta'III turn over Ser11-Gly12 and a helical turn from Gly24 to Cys28 are identified. This structure is very similar to the equivalent regions of the X-ray structure of wheat germ agglutinin and the NMR structure of hevein.
Martins JC, Maes D, Loris R, Pepermans HA, Wyns L, Willem R, Verheyden P. H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus. J Mol Biol. 1996 May 3;258(2):322-33.
H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus.

It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events.

Muraki M.
The importance of CH/pi interactions to the function of carbohydrate binding proteins.
Protein Pept Lett. 2002 Jun;9(3):195-209.

The importance of CH/pi interactions to the function of carbohydrate binding proteins.

HEV32, a 32-residue, truncated hevein lacking eleven C-terminal amino acids, was synthesized by solid-phase methodology and correctly folded with three cysteine bridge pairs. The affinities of HEV32 for small chitin fragments--in the forms of N,N',N-triacetylchitotriose ((GlcNAc)3) (millimolar) and N,N',N,N',N,N'-hexaacetylchitohexaose ((GlcNAc)6) (micromolar)--as measured by NMR and fluorescence methods, are comparable with those of native hevein. The HEV32 ligand-binding process is enthalpy driven, while entropy opposes binding. The NMR structure of ligand-bound HEV32 in aqueous solution was determined to be highly similar to the NMR structure of ligand-bound hevein. Solvated molecular-dynamics simulations were performed in order to monitor the changes in side-chain conformation of the binding site of HEV32 and hevein upon interaction with ligands. The calculations suggest that the Trp21 side-chain orientation of HEV32 in the free form differs from that in the bound state; this agrees with fluorescence and thermodynamic data. HEV32 provides a simple molecular model for studying protein-carbohydrate interactions and for understanding the physiological relevance of small native hevein domains lacking C-terminal residues.

Aboitiz N, Vila-Perello M, Groves P, Asensio JL, Andreu D, Canada FJ,
Jimenez-Barbero J.
NMR and modeling studies of protein-carbohydrate interactions: synthesis,
three-dimensional structure, and recognition properties of a minimum hevein
domain with binding affinity for chitooligosaccharides.
Chembiochem. 2004 Sep 6;5(9):1245-55.

Chembiochem : a European journal of chemical biology
NMR and modeling studies of protein-carbohydrate interactions: synthesis, three-dimensional structure, and recognition properties of a minimum hevein domain with binding affinity for chitooligosaccharides.

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.

Asensio JL, Siebert HC, von Der Lieth CW, Laynez J, Bruix M, Soedjanaamadja UM,
Beintema JJ, Canada FJ, Gabius HJ, Jimenez-Barbero J.
NMR investigations of protein-carbohydrate interactions: studies on the
relevance of Trp/Tyr variations in lectin binding sites as deduced from
titration microcalorimetry and NMR studies on hevein domains. Determination of
the NMR structure of the complex between pseudohevein and
N,N',N-triacetylchitotriose.
Proteins. 2000 Aug 1;40(2):218-36.

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose.

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.

Asensio JL, Canada FJ, Siebert HC, Laynez J, Poveda A, Nieto PM, Soedjanaamadja
UM, Gabius HJ, Jimenez-Barbero J.
Structural basis for chitin recognition by defense proteins: GlcNAc residues
are bound in a multivalent fashion by extended binding sites in hevein domains.
Chem Biol. 2000 Jul;7(7):529-43.

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.

The secondary structure of this protein presents beta-sheet on residues over
residues 13 to 23.
This protein is a hevein domain, and has high homology with AcAMP2
antimicrobial peptide. Carbohydrate binding site involves residues S16, Nal18,
Y20 and Y27.

A Chain A, 30 Nmr Structures Of Acamp2-LikePeptide With Non Natural Beta-(2-Naphthyl)-AlanineResidue

The residue Phe18 of native AcAMP2 was changed to beta-(2-naphtyl)-alanine. The aminoacid was manually assembled by solid phase synthesis using Fmoc chemistry according to satandard protocols.

The software from Bruker performs acquisition and processing of NMR experiments.

This program helps visualization and assignment of 2D NMR spectra.
Bartels C., Xia T., Billeter M., Guntert P. and Wuthrich K. (1995)
J. Biol. NMR 6, 1-10.
The program XEASY for computer-supported NMR spectral analysis of biological
macromolecules.

This program yields a collection of protein structures that fit the 1H-1H
distance constraints experimentally obtained.
Guntert P., Mumenthaler C. and Wuthrich K. (1997)
J. Mol. Biol. 273, 283-298.
Torsion angle dynamics for NMR structure calculation with the new program DYANA.

The programs used from this package perform molecular dynamics calculations on
protein structures. They also convert dyana-format pdb files into amber-format
pdb files.
Pearlman D. A., Case D. A., Caldwell J. W., Cheatham T. E., DeBolt S., Ross W.
S., Ferguson D., Seibel G. L., and Kollman P. A. (1995)
Comp. Phys. Commun. 91, 1-41.
AMBER, a package of computer programs for applying molecular mechanics, normal
mode analysis, molecular dynamics and free energy calculations to simulate the
structural and energetic properties of molecules.