TY - JOUR
T1 - Chimeric approach for narrowing a membrane-inserting region within human perforin
AU - Neely, Amy E.
AU - Mandigo, Kimberly A.
AU - Robinson, Rebekah L.
AU - Ness, Traci L.
AU - Weiland, Mitch H.
N1 - Publisher Copyright:
© The Author 2016. Published by Oxford University Press.
PY - 2017/2/1
Y1 - 2017/2/1
N2 - Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved β-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane β-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.
AB - Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved β-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane β-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.
KW - MACPF
KW - TMH
KW - perforin
KW - perfringolysin-O
KW - pore formation
UR - http://www.scopus.com/inward/record.url?scp=85021099783&partnerID=8YFLogxK
U2 - 10.1093/protein/gzw069
DO - 10.1093/protein/gzw069
M3 - Article
C2 - 27980121
AN - SCOPUS:85021099783
SN - 1741-0126
VL - 30
SP - 105
EP - 111
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 2
ER -