In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein.
Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long alpha-helix of residues 30-44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure-function studies of plant LRR proteins.
Kolade
Olatomirin O
OO
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Bamford
Vicki A
VA
Ancillo Anton
Gema
G
Jones
Jonathan D G
JD
Vera
Pablo
P
Hemmings
Andrew M
AM
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D066298:In Vitro Techniques; D007930:Leucine / Q000737:chemistry*; D000090263:Leucine-Rich Repeat Proteins; D018551:Solanum lycopersicum / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
D004220:Disulfides; D000090263:Leucine-Rich Repeat Proteins; D010940:Plant Proteins; D011506:Proteins; D014364:Tryptophan; D005982:Glutathione Transferase; D007930:Leucine; D003545:Cysteine