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 MyxovirinTyler 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

∆ p

pm

Residue

11 10 9 8 7 61H (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

****