Tài liệu Báo cáo khoa học: Key role of the loop connecting the two beta strands of mussel defensin

Key role of the loop connecting the two beta strands of mussel

defensin in its antimicrobial activity

Bernard Romestand1

, Franck Molina2

, Ve´ronique Richard1

, Philippe Roch1 and Claude Granier2

1

DRIM, Universite´ Montpellier 2, France; 2

Centre de Biotechnologie et Pharmacologie pour la Sante´, CNRS UMR Montpellier,

France

To elucidate the structural features of the mussel defensin

MGD1 required for antimicrobial activity, we synthesized a

series of peptides corresponding to the main known secon￾dary structures of the molecule and evaluated their activity

towards Gram-positive and Gram-negative bacteria, and

filamentous fungi. We found that the nonapeptide corres￾ponding to residues 25–33 of MGD1 (CGGWHRLRC)

exhibited bacteriostatic activity once it was cyclized by a

non-naturally occurring disulfide bridge. Longer peptides

corresponding to the amino acid sequences of the a-helical

part or to the b-strands ofMGD1 had no detectable activity.

The bacteriostatic activity of the sequence 25–33 was strictly

dependent on the bridging of Cys25 and Cys33 and was

proportional to the theoretical isoelectric point of the pep￾tide, as deduced from the variation of activity in a set

of peptide analogues of the 25–33 sequence with different

numbers of cationic charges. By using confocal fluorescence

microscopy, we found that the cyclic peptides bound to

Gram-positive bacteria without apparent lysis. However, by

using a fluorescent dye, we observed that dead bacteria

had been permeated by the cyclic peptide 25–33. Sequence

comparisons in the family of arthopod defensins indicate

that MGD1 belongs to a subfamily of the insect defensins,

characterized by the constant occurrence of both positively

charged and hydrophobic amino acids in the loop. Model￾ling studies showed that in the MGD1 structure, positively

charged and hydrophobic residues are organized in two

layered clusters, which might have a functional significance

in the docking of MGD1 to the bacterial membrane.

Keywords: defensin; antimicrobial peptide; solid-phase syn￾thesis; active loop; cyclic peptide.

Antimicrobial peptides are essential actors of innate immu￾nity that have been conserved throughout evolution. Many

such molecules have been purified over the past decade,

from vertebrates, invertebrates, plants and bacteria. Some

of these compounds have been investigated with a view to

possible therapeutic use [1], as an alarming increase of

resistance of microorganisms to classical antibiotics has

been reported [2,3]. Defensins are antimicrobial peptides

isolated from mammals [4], arthropods [5,6], plants [7,8]

and more recently from molluscs [9,10]. They are cationic

molecules belonging to the cysteine-rich family of anti￾microbial peptides. Mammalian defensins comprise human

neutrophil peptides (HPN-1–4), human defensins (HD-5

and 6), two human b defensins (HBD-1 and 2) [11–13] and a

cyclic rhesus theta defensin (RTD-1) [14]. Although all

defensins display antibacterial activity, mammalian and

other vertebrate defensins are quite different from the

arthropod/mollusc defensins in terms of both sequence and

structure [15–17].

MGD1 is a defensin of 39 residues, which has been

isolated from plasma and haemocytes of the edible Medi￾terranean mussel, Mytilus galloprovincialis [10,18]. MGD1

shares the so-called cysteine-stabilized alpha-beta motif

(Csab) with arthropod defensins [19], but it is characterized

by the presence of an additional disulfide bond. The three￾dimensional solution structure of MGD1 has been estab￾lished using 1

H-NMR and mainly consists of a helical part

(residues 7–16) and two antiparallel b-strands (residues

20–25 and 33–39) [16]. The a-helix and the b1-strand are

connected by a distorted type II turn (loop 2), whereas the

loop connecting both strands of the b-sheet (residues 25–33)

includes a type III¢ turn (loop 3) and points out of the core

of the protein.

There is a consensus view that defensins act by disrupting

the cytoplasmmembrane[20–24], although the exactmode of

action is not clearly established. To gain further insight into

the structural requirements for antimicrobial activity, we

designed a number of peptide fragments based on the

knowledge of the structure ofMGD1[16]. Synthetic peptides,

including amino acid substitutions, were tested for bacterio￾static activity and revealed the crucial role of loop 3 and the

effect of positive charges. Loop 3-derived peptides were

found to bind to Gram-positive bacteria resulting in

permeation of the membrane and bacterial killing.

Materials and methods

Synthesis of soluble peptides

All soluble peptides were synthesized on an Abimed AMS

422 synthesizer by Fmoc chemistry [25,26]. Peptides were

deprotected and released from the Rink amide resin

Correspondence to P. Roch, Laboratoire DRIM, CC080, Universite´

Montpellier 2, Place E. Bataillon, 34095 Montpellier, France.

Fax: + 33 4 67 14 46 73, Tel.: + 33 4 67 14 47 12,

E-mail: [email protected]

Abbreviations: MGD, Mytilus galloprovincialis defensin;

MIC, minimal inhibitory concentration.

(Received 14 February 2003, revised 28 April 2003,

accepted 08 May 2003)

Eur. J. Biochem. 270, 2805–2813 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03657.x