Residues Phe18 and Tyr20 have been mutated to 4-fluorophenylalanine in the
molecule studied here

The atom HA2 of GLY22 shows up at 1.828; due the proximity to the aromatic
system of the residue 27

Information obtained from PDB's Chemical Component Dictionary
at http://wwpdb-remediation.rutgers.edu/downloads.html
Downloaded on Mon Aug 8 14:18:20 2011

(2S)-2-amino-3-(4-fluorophenyl)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. Review.

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, F18FF,
F20FF and Y27.

18 Nmr Structures Of Acamp2-Like Peptide With Non Natural Fluoroaromatic Residue (Acamp2f18pffY20PFF) COMPLEX WITH N,N,N- Triac

The residues Phe18 and Tyr20 of the native AcAMP2 were changed to
4-fluorophenylalanine. The aminoacids were manually assembled by solid phase
synthesis using Fmoc chemistry according to satandard protocols.

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

The structures are based on a total 314 cross peaks, 248 NOE-derived distance restraints, and finally 208 distance constraints and 18 come from cys-cys disulfide were used in the final round of calculation

The software performs acquisition and processing of NMR experiments

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 helps visualization and assignment of 2D NMR spectra.

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.
This program yields a collection of protein structures that fit the 1H-1H
distance constraints experimentally obtained.

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.
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.