Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.
KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.
Structural Biology & Bio-Informatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mallick Road, Kolkata 700032, India.
Achinta
A
Sannigrahi
Department of Physics, Jadavpur University , 188, Raja S. C. Mallick Road, Kolkata 700032, India.
Pabitra
P
Maity
Department of Physics, Jadavpur University , 188, Raja S. C. Mallick Road, Kolkata 700032, India.
Sanat
S
Karmakar
Structural Biology & Bio-Informatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mallick Road, Kolkata 700032, India.
Krishnananda
0000-0002-1449-8909
K
Chattopadhyay
D002784:Cholesterol / Q000378:metabolism*; D006790:Host-Parasite Interactions; D006801:Humans; D007891:Leishmania / Q000737:chemistry / Q000235:genetics / Q000469:parasitology / Q000502:physiology*; D007896:Leishmaniasis / Q000378:metabolism* / Q000469:parasitology; D008264:Macrophages / Q000378:metabolism / Q000469:parasitology; D008958:Models, Molecular; D010743:Phospholipids / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010743:Phospholipids; D015800:Protozoan Proteins; D014364:Tryptophan; D002784:Cholesterol