Insight into Structure and Biological Functions
5. Structure of the Biologically Active Peptides of Lf
It was originally thought that the antimicrobial activity of Lf was only dependent on its ability to
2007 ). Another example of complex formation by Lf, this time with ceruloplasmin, has been localized to the N - lobe and may involve this region (Sabatucci et al. 2007 ). Recently, another region of the N1 region of Lf encompassing residues 265 – 284 of the bovine protein (and its equivalent in other Lfs), called Lfampin, was shown to be essential in the antimicrobial activities of the molecule (van der Kraan et al. 2004, 2006 ). As shown in Table 13.1 , the Lfampin sequence is close to the Lfcin domain.
The cationic charges at its C - terminal end contribute to the antimicrobial activity of Lf, while its N - terminal end forms an amphipathic helix (van der Kraan, Nazmi et al. 2005 ; van der Kraan, van der Made et al. 2005 ). Lastly, it should be mentioned receptor – binding sites (Legrand et al. 1992 ); and
residues 1 – 31 as a DNA - binding site (van Berkel et al. 1997 ).
The fi rst helix (residues 12 – 31 in hLf) forms the major part of the bactericidal domain identifi ed by Bellamy et al. (Bellamy et al. 1992 ), described as the Lfcin domain (Gifford et al. 2005 ) (Figure 13.2 ).
Recent results show that this bactericidal domain, which is a highly exposed feature of the Lf surface, can also serve as a macromolecular binding surface.
A recent crystallographic analysis shows that the N - lobe of hLf binds specifi cally through this region to a bacterial cell surface protein, the pneumococcal surface protein A (PspA) from the important human pathogen Streptococcus pyogenes (Senkovich et al.
Figure 13.2. Localization of the Lfcin domain and Lfampin sequence on Lf. The fi gure shows the front (A and C) and left side (B and D) views of the Lf molecule (pdb reference 2BJJ) with the positions of LfcinH (black and light grey), LfcinB (light grey), and LfampinB or LfampinH (dark grey) (Bellamy et al. 1992 ; van der Kraan et al. 2004 ) . Ribbon foldings of the peptide domains are shown in A and B, and the solvent - accessible surface of the molecule is shown in C and D. Molecular modeling was performed using the Chimera software ( http://www.cgl.ucsf.edu/chimera/ ).
The β - sheet gives the peptide an amphipathic struc- ture, with most of the hydrophobic residues residing on one side of the β - sheet plane, while the majority of the hydrophilic residues rest on the other side.
The LfcinB structure in solution at low salt concen- tration contrasts the conformation of the peptide in the Lf (Figure 13.3 ). In the crystal structure, an that a serine protease activity has been detected in
the N - terminal lobe of Lf that could play a role in its antibacterial activity against Hemophilus spp. K 73 and S 259 of hLf are essential residues for the protease activity (Hendrixson et al. 2003 ).
5.2. Structure of Lfcin
Antimicrobial peptides encompass a wide variety of structures, of which many have α - helical motifs.
The majority of these peptides are cationic and amphipathic, but there are also hydrophobic α - helical peptides that possess antimicrobial activity, and there are antimicrobial peptides that are rich in a certain specifi c amino acid such as Trp or His (Chan et al. 2006 ). In spite of the structural diver- sity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic struc- ture that allows them to bind to the membrane interface.
Limited proteolysis of Lf by trypsin leads to the release of the N - t and C - t lobes and the N - 2 domain (Legrand et al. 1984 ), while hydrolysis of Lf by pepsin yields the Lfcin peptide (Bellamy et al.
1992 ). Bovine Lfcin (LfcinB) has been investigated most extensively since it is the most active Lfcin found across various species and also the most readily available (reviewed in Gifford et al. 2005 ).
The Lfcin peptides from different species display drastic sequence differences but still share some similarities. LfcinB encompasses 25 residues, including 2 Trp residues at positions 6 and 8 and a disulfi de bond between its 2 cysteine residues (Table 13.1 ; Figure 13.3 ).
The structure of LfcinB in aqueous solution was solved by homonuclear NMR and shows that the peptide adopts a short, twisted, antiparallel β - sheet conformation (Figure 13.3 ) (Hwang et al. 1998 ).
Table 13.1. Sequences of the Lfcin and Lfampin peptides from h L f and b L f .
Peptide Sequence No. of aa Net charge
LfcinH GRRRRSVQWCAVSQPEATKC - FQWQRNMRKVRGPPVSCIKR - DSPIQCIQA 49 +9
LfcinB FKCRRWQWRMKKLGAPSITCVRRAE 25 +8
LfampinH WNLLRQAQEKFGKDKSP 17 +2
LfampinB WKLLSKAQEKFGKNKSR 17 +5
Figure 13.3. Structure of LfcinB. The fi gure shows the ribbon foldings of (A) the LfcinB domain in bLf (residues 17 – 41) as determined by x - ray diffraction (pdb reference 1BLF) (Moore et al. 1997 ) , and (B) the LfcinB 25 - amino - acid peptide in aqueous solution as determined by NMR spectroscopy (pdb reference 1LFC) (Hwang et al. 1998 ) . The positions of the positively charged amino acid residues are indicated, as well the positions of the Trp residues 19 and 21 in bLf. Molecular modeling was performed using the Chimera software ( http://
www.cgl.ucsf.edu/chimera/ ).
was never evidenced as a free peptide in secretions, although its release from the whole protein was demonstrated using a one - step proteolysis protocol with AsnP protease from Pseudomonas fragi (Bolscher et al. 2006 ) .
Recently, human Lfampin (LfampinH) peptides corresponding to residues 269 – 285 in hLf were synthesized and characterized using NMR spec- troscopy, fl uorescence, and differential scanning calorimetry to determine which residues were important for the antimicrobial activity against Candida albicans (Haney et al. 2009 ). The authors concluded that the amphipathic N - terminal α - helix appears to be responsible for mediating the interac- tion between the peptide and the phospholipid bilayer, but the antimicrobial activity of the LfampinH peptides is dictated by the net cationic charge of the fl exible C - terminal region.
6. Biological Roles of Lf