Limits...
Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface.

Matson JS, Nilles ML - BMC Microbiol. (2002)

Bottom Line: Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction.Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif.We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA. jyl_matson@und.nodak.edu

ABSTRACT

Background: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

Show MeSH

Related in: MedlinePlus

Pairwise BLAST alignment of LcrG and PcrG. Amino acid sequence alignment of LcrG from Y. pestis (NCBI RefSeq, NP_395166; ) and PcrG from P. aeruginosa (NCBI RefSeq, NP_250396; ). Overall the two proteins are 43% identical and 56% similar. The sequences were aligned using the BLAST algorithm as implemented for pairwise alignments [37]. The region highlighted in yellow (amino acids 7–40) is the smallest region identified that interacts with LcrV. The region in blue (amino acids 39–53) corresponds to a previously described deletion that eliminated secretion blocking activity [5]. The amino acids identified with red asterisks are residues identified as participating in the LcrG-LcrV interaction (A16, S23, and L30). The residue marked with the blue asterisk (F48) is required for secretion blocking activity of LcrG.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC117220&req=5

Figure 2: Pairwise BLAST alignment of LcrG and PcrG. Amino acid sequence alignment of LcrG from Y. pestis (NCBI RefSeq, NP_395166; ) and PcrG from P. aeruginosa (NCBI RefSeq, NP_250396; ). Overall the two proteins are 43% identical and 56% similar. The sequences were aligned using the BLAST algorithm as implemented for pairwise alignments [37]. The region highlighted in yellow (amino acids 7–40) is the smallest region identified that interacts with LcrV. The region in blue (amino acids 39–53) corresponds to a previously described deletion that eliminated secretion blocking activity [5]. The amino acids identified with red asterisks are residues identified as participating in the LcrG-LcrV interaction (A16, S23, and L30). The residue marked with the blue asterisk (F48) is required for secretion blocking activity of LcrG.

Mentions: LcrG and LcrV physically interact in Y. pestis [4], and that interaction plays a significant role in the regulation of Yops secretion [20]. LcrG and LcrV interact in a yeast two-hybrid assay [20], therefore this technique was used to further investigate the region of LcrG required for its interaction with LcrV. Truncations of LcrG were constructed and fused to the GAL4 activation domain of pACT2. These plasmids were co-transformed into yeast strain Y187 with full-length LcrV fused to the GAL4 DNA binding domain of plasmid pAS2-1 to determine if the LcrG truncations could continue to participate in an interaction with LcrV. Using a filter-lift assay as a qualitative determination of an interaction and a liquid β-galactosidase assay to quantify the results, we found that the LcrV-interaction domain of LcrG lies in the N-terminus of the protein. The smallest linear region of LcrG that was still capable of interacting with LcrV was determined to be amino acids 7–40 (Fig. 1; shown highlighted in yellow in Fig. 2). Deletion of five additional amino acids from the N-terminus of the 7–40 construct or seven amino acids from the C-terminus of the 7–40 construct eliminated our ability to detect an interaction using this system (Fig. 1). Interestingly, this approximate region of LcrG (amino acids 7–35) is predicted to form a coiled-coil domain when LcrG is analyzed using the COILS algorithm [26] (data not shown). These results suggest that the LcrG-LcrV interaction requires the N-terminus of LcrG and may involve a coiled-coil domain.


Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface.

Matson JS, Nilles ML - BMC Microbiol. (2002)

Pairwise BLAST alignment of LcrG and PcrG. Amino acid sequence alignment of LcrG from Y. pestis (NCBI RefSeq, NP_395166; ) and PcrG from P. aeruginosa (NCBI RefSeq, NP_250396; ). Overall the two proteins are 43% identical and 56% similar. The sequences were aligned using the BLAST algorithm as implemented for pairwise alignments [37]. The region highlighted in yellow (amino acids 7–40) is the smallest region identified that interacts with LcrV. The region in blue (amino acids 39–53) corresponds to a previously described deletion that eliminated secretion blocking activity [5]. The amino acids identified with red asterisks are residues identified as participating in the LcrG-LcrV interaction (A16, S23, and L30). The residue marked with the blue asterisk (F48) is required for secretion blocking activity of LcrG.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117220&req=5

Figure 2: Pairwise BLAST alignment of LcrG and PcrG. Amino acid sequence alignment of LcrG from Y. pestis (NCBI RefSeq, NP_395166; ) and PcrG from P. aeruginosa (NCBI RefSeq, NP_250396; ). Overall the two proteins are 43% identical and 56% similar. The sequences were aligned using the BLAST algorithm as implemented for pairwise alignments [37]. The region highlighted in yellow (amino acids 7–40) is the smallest region identified that interacts with LcrV. The region in blue (amino acids 39–53) corresponds to a previously described deletion that eliminated secretion blocking activity [5]. The amino acids identified with red asterisks are residues identified as participating in the LcrG-LcrV interaction (A16, S23, and L30). The residue marked with the blue asterisk (F48) is required for secretion blocking activity of LcrG.
Mentions: LcrG and LcrV physically interact in Y. pestis [4], and that interaction plays a significant role in the regulation of Yops secretion [20]. LcrG and LcrV interact in a yeast two-hybrid assay [20], therefore this technique was used to further investigate the region of LcrG required for its interaction with LcrV. Truncations of LcrG were constructed and fused to the GAL4 activation domain of pACT2. These plasmids were co-transformed into yeast strain Y187 with full-length LcrV fused to the GAL4 DNA binding domain of plasmid pAS2-1 to determine if the LcrG truncations could continue to participate in an interaction with LcrV. Using a filter-lift assay as a qualitative determination of an interaction and a liquid β-galactosidase assay to quantify the results, we found that the LcrV-interaction domain of LcrG lies in the N-terminus of the protein. The smallest linear region of LcrG that was still capable of interacting with LcrV was determined to be amino acids 7–40 (Fig. 1; shown highlighted in yellow in Fig. 2). Deletion of five additional amino acids from the N-terminus of the 7–40 construct or seven amino acids from the C-terminus of the 7–40 construct eliminated our ability to detect an interaction using this system (Fig. 1). Interestingly, this approximate region of LcrG (amino acids 7–35) is predicted to form a coiled-coil domain when LcrG is analyzed using the COILS algorithm [26] (data not shown). These results suggest that the LcrG-LcrV interaction requires the N-terminus of LcrG and may involve a coiled-coil domain.

Bottom Line: Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction.Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif.We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA. jyl_matson@und.nodak.edu

ABSTRACT

Background: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

Show MeSH
Related in: MedlinePlus