Characterization of the Interaction of Yersinia Type

III Secretion System Chaperone LcrG to Tip

Protein LcrV Mason Wilkinson

Schraidt et al. PLoS Pathog 6(4), 2010

Outline

• Introduction to the Type III Secretion System• My Project and its Purpose• Methods• Results• Future Directions• Questions

Gram-Negative Bacteria Require the T3SS for Infection

Bacteria Associated Disease

Yersinia pestis Bubonic Plague

Shigella flexneri Dysentery

Burkholderia pseudomallei Melioidosis

Salmonella typhimurium Salmonellosis

Pseudomonas aeruginosa Pneumonia

Chlamydia trachomatis Trachoma and STD

Escherichia coli Gastroenteritis

These Gram-negative bacteria have become resistant to current antibiotics.

Wang et al, Mol. BioSyst., 2008

LcrVType III Secretion System

• The Tip Protein LcrV is essential for T3SS function.• The role of LcrV is to detect the host environment and regulate

secretion.• Chaperone LcrG prevents premature assembly of the T3SS apparatus.

Why Use Nuclear Magnetic Resonance?

• LcrV and LcrG do not co-crystalize.• NMR provides residue-specific information.

Isoleucine

Isoleucine Methyl Probes for NMR

Isotopically-labeled alpha ketoacids are used to label isoleucine delta methyls with 13C.

NMR Spectrum of Isoleucine Methyl-Labeled LcrV

1 NMR Peak = 1 Isoleucine Methyl

Isoleucine Methyl Assignment by Site-Directed Mutagenesis

1. Introduce IL point mutant via Polymerase Chain Reaction (PCR).

2. Transform mutant plasmid into E. coli.

3. Express Mutant LcrV.

Thermo Fisher Scientific. ”Phusion Site-Directed Mutagenesis Protocol.”

Making an isotopically labeled recombinant protein for NMR Plasmid containing protein sequence +E. coli BL21(DE3) in minimal media

Grow cells, add α-ketoacids (Isoleucine methyl labels)and induce expression with IPTG

Purification by affinity chromatography

Concentrate protein andtransfer to NMR tube

Acquire data using 800 MHz magnet

LcrV

Assignment of Isoleucine 206 by Site Directed Mutagenesis

Complete Assignment of LcrV Isoleucine Methyls

LcrG interacts with specific isoleucines of LcrV.

Kaur, Kawaljit. 2014-2016.

The LcrV Surface Affected by LcrG

Conclusions

• PCR was used to introduce point mutations into LcrV and mutant proteins were expressed and purified for NMR.

• All 25 isoleucine methyl NMR peaks of LcrV were assigned.

• These isoleucine methyl assignments were used to map the LcrV surface affected by LcrG.

Future Directions

• Publish results in the journal, Biochemistry.

• Use our isoleucine methyl assignments to test for interaction between LcrV and small molecules to develop T3SS inhibitors.

AcknowledgementsDe Guzman Lab:Dr. Roberto N. De GuzmanKawaljit KaurDr. Supratim DeyAndrew McShanPallavi Guha BiswasAmritanshu ChakravartySanjay YadavaSikta Patnaik (Former KU)

Funding:

KU Biotechnology Training Grant:T32-GM008359

NIH Grant:R01-AI074856

Biomolecular NMR KU Core:P30-GM110761

Yersinia pestis says… Thank you for your attention!

15N Amide-Labeled Wild Type LcrV

Lactose

Isopropyl-beta-ThiogalactosideNamrata. Biochemistry for Medics. 2016.

LcrV I309L – First Purification LcrV I309L – After TEV Cleavage

Bears HIS-TagNo HIS-Tag

LcrVHIS6

TEV Cleavage

Site

HIS-Tag

Imidazole

KPL. “HIS-Tagged Protein Purification” Protocol. 2016.