PDBsum entry 2nlc

Antimicrobial protein

PDB id

2nlc

Contents

			Protein chain

					36 a.a.

			Ligands

					SO4 ×6


					ACT

			Waters ×214

References listed in PDB file

Key reference

Title

Studies of the biological properties of human beta-Defensin 1.

Authors

M.Pazgier, A.Prahl, D.M.Hoover, J.Lubkowski.

Ref.

J Biol Chem, 2007, 282, 1819-1829. [DOI no: 10.1074/jbc.M607210200]

PubMed id

17071614

Abstract

Defensins are small (30-45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and fungi, some enveloped viruses, and other activities such as chemoattraction of a range of different cell types to the sites of inflammation. These proteins represent attractive targets for developing novel antimicrobial agents and modulators of immune responses with therapeutic applicability. In this report, we present the results of functional and structural studies of 26 single-site mutants of human beta-defensin 1 (hBD1). All mutants were assayed for antimicrobial activity against Escherichia coli (ATCC strain 25922) and for chemotactic activity with CCR6-transfected HEK293 cells. To analyze the structural implications of mutagenesis and to verify the correctness of the disulfide connectivity, we used x-ray crystallography to conduct complete structural studies for 10 mutants in which the topology of disulfides was the same as in the native hBD1. Mutations did not induce significant changes of the tertiary structure, suggesting that the observed alterations of biological properties of the mutants were solely associated with changes in the respective side chains. We found that cationic residues located near the C terminus (Arg(29), Lys(31), Lys(33), and Lys(36)) of hBD1 define most of the anti-E. coli in vitro activity of this protein. In turn, nearly all mutations altering the CCR6-mediated chemotaxis are located at one area of the protein, defined by the N-terminal alpha-helical region (Asp(1)... Ser(8)) and a few topologically adjacent residues (Lys(22), Arg(29), and Lys(33)). These experimental results allow for the first time drafting of the CCR6-epitope for a defensin molecule.



	Figure 2. FIGURE 2. Structural alignment of hBD-1 mutants and hBD-1 wild type. C[ ]traces of six independent monomers of hBD-1 wild type (35) (red) were superimposed on four monomers of the N04A mutant (green), four monomers of S08A mutant (blue), two monomers of Q11A mutant (magenta), two monomers of L13A mutant (cyan), two monomers of L13E mutant (orange), four monomers of K22E mutant (gray), four monomers Q24A (black), four monomers of Q24E mutant (yellow), four monomers of K31A mutant (violet), and one monomer of K31E mutant (pink). Ribbon representation of the secondary structure of hBD-1 wild type is shown in the top right panel. The figure was prepared with program PyMOL (pymol.org).		Figure 5. FIGURE 5. Surface distribution of the residues important for antibacterial activity of hBD1. The electrostatic potential, mapped on the solvent-accessible surface, is shown in the upper panel. Contributions to the potential by the positively charged residues are shown in blue, while the contributions by the anionic residues are marked in red. The lower panel shows the distribution of the residues identified as important for the anti-E. coli activity of hBD1. The area of the molecule that, upon mutation, led to increased activity is shown in green. The residues found as determinants for high anti-E. coli activity (RA[50] of their mutants was <0.11) are indicated in red (for definition of RA[xx], see Table 4). The residues identified as moderately important for anti-E. coli activity (RA[50] >0.3 and RA[90] <0.11) are marked in yellow. The ribbon diagram of the hBD1 monomer with side chains of all the residues identified here as contributing to antibacterial activity of this protein is also shown for reference. All objects are in equivalent or rotated by 180° orientations. The figures were prepared with programs PyMOL (pymol.org) and Grasp (53).

PROCHECK

	Headers