This document discusses the lectin Myxovirin, which is similar to the lectin Scytovirin. It summarizes that Myxovirin binds to the sugar Man9, and the authors have assigned resonances in the NMR spectrum of Myxovirin. The authors aim to determine the solution structure of Myxovirin when bound and unbound to Man4, and to characterize chimeric proteins that fuse Myxovirin to antifungal peptides.

1. Myxovirin Man9 Binding and Structure
Each domain of Scytovirin is known
to specifically bind Man4 (Figure 4),
the D3 arm of Man9. We have also
observed that Myxovirin binds Man9.
For Man9 titration experiments,
Mannonanose-di-(N-acetyl-D-glucos-
amine) is purified from soy flour. Soy
bean agglutinin (SBA) is separated
from soy flour on a sepharose-N-
caproyl-galactosamine affinity
column, and the SBA is digested with
pronase, releasing (Man)9(GlcNAc)2.
The Man9 is then purified on a sizing
column.
Future Aims
• Solve the solution structure of Myxovirin
• Solve the structure of Myxovirin bound to Man4
• Construct and characterize high mannose-binding
lectin/antifungal peptide chimera
To better understand the structure/function relationship of high
mannose-binding lectins, we aim to solve the structure of Myxovirin with
and without Man4. We currently have NOESY spectra and are working
through them to produce a solution structure. We are also investigating
the potential of lectin/antifungal peptide chimeric proteins. These
constructs should increase the effectiveness of the antifungal properties
of high mannose-binding lectins. Scytovirin and Myxovirin are being
fused to common antifungal peptides by variously sized flexible linkers.
The ability of Myxovirin to withstand N-terminal modifications may allow
us to link multiple antifungal peptides on the termini. Small antifungal
peptides that are being studied include dermaseptins, cecropins, and
other small, α-helical, membrane disrupting peptides.
Antifungal
Peptide 1
Antifungal
Peptide 2
Myxovirin
Novel Antimicrobial Lectin Myxovirin
Tyler H. Jones* and Dr. Robert L. McFeeters
Department of Chemistry, The University of Alabama in Huntsville, *Email: thj0002@uah.edu
Comprehensive chemical shift assignments of Myxovirin have been
completed. Shown in the 1H-15N HSQC spectrum (Figure 2), 35 of 38
non-proline backbone amide resonances have been assigned. With the
exception of Gly1, all backbone carbonyl resonances and all backbone
Hα and Cα resonances are assigned. Sequential assignments were
carried out using a 0.5 mM 15N- and 13C/15N-labeled sample. Spectra
acquired include 15N-HSQC, HNCACB, CBCA(CO)NH, HNCO,
HN(CA)CO, H(CCO)NH, (H)CC(CO)NH, and HCCH-TOCSY. Of note,
similar to Scytovirin, a comparison of Cα chemical shift values to
random coil values shows no regions of extended regular secondary
structure (Figure 3).
Introduction
High mannose-binding lectins have long been studied for their potent
antiviral activity. Among these lectins, Scytovirin, a small 9.5 kDa
protein from the cyanobacterium Scytonema varium, is distinctive for its
lack of extended secondary structure, potent antiviral efficacy, and
benign safety profile. Recently, our lab has extended the application of
Scytovirin inhibition to numerous pathogenic fungi including
Cryptococcus neoformans. Through broth macrodilution testing, we
found minimum inhibitory concentration values in the sub-micromolar
range. To further understand how this lectin inhibits fungi, we searched
for proteins similar to Scytovirin. A Scytovirin-like domain, Myxovirin,
was found between an N-terminal signal peptide and beta-trefoil motif
similar to that of the Ricin B chain in the genome of the proteobacteria
Myxococcus fulvus. Myxovirin shares 72% amino acid sequence
identity (28 of 39 identical residues) and 87% homology to the C-
terminal carbohydrate binding domain of Scytovirin.
Figure 1: Native protein arrangement from Myxococcus fulvus (Top).
Sequence alignment of the carbohydrate binding domains of Scytovirin,
SD1 and SD2 (red), with Myxovirin (blue). Boxes indicate aromatic
residues involved in carbohydrate binding and asterisks indicate cysteines
participating in intradomain disulfide bonds (Bottom).
While Scytovirin has two similar carbohydrate binding domains,
Myxovirin has a single domain. Scytovirin has previously shown to be
intolerant of N-terminal alterations. Conversely, native Myxovirin was
found with an N-terminal signal peptide, indicating that it is capable of
tolerating these modifications. Of note, Myxovirin conserves the distinct
intradomain disulfide bonding cysteines and positions of aromatic
residues found to be important for carbohydrate binding in Scytovirin.
The single domain of Myxovirin allows for solution structure
determination and solving of the bound structure.
Figure 2: 500 MHz 1H-15N-HSQC of 15N labeled Myxovirin at pH 5.5, 298 K.
NMR Resonance Assignments of Myxovirin
Figure 3: Chemical shift index plot of the difference between measured Ca
resonances and their random coil values for each residue indicates no
regions of extended secondary structure.
Figure 4: Man4
Manα(1-2)Manα(1-6)
Manα(1-6)Man
Figure 5: Chimeric construct of Myxovirin with N- and C-terminal
antifungal peptides.
1 6 11 16 21 26 31 36 41
~
4
-4
-2
2
0
∆ppm
Residue
11 10 9 8 7 6
1H (ppm)
110
120
130
15N(ppm)Acknowledgements The authors would like to thank the members of the McFeeters Lab, Dr. Hana McFeeters (UAH), and Dr. Kevin Anderson (Oakwood University)
Signal Peptide Myxo Ricin B chain
40 80 91 250
SD1
SD2
Myxo
****