Using mutagenesis and structural biology to map the binding site for the Plasmodium falciparum merozoite protein PfRh4 on the human immune adherence receptor.
To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.
From the Washington University School of Medicine, Division of Rheumatology, Department of Internal Medicine, St. Louis, Missouri 63110.
Hyon Ju
HJ
Park
Mara
M
Guariento
Mateusz
M
Maciejewski
Richard
R
Hauhart
Wai-Hong
WH
Tham
Alan F
AF
Cowman
Christoph Q
CQ
Schmidt
Haydyn D T
HD
Mertens
M Kathryn
MK
Liszewski
Dennis E
DE
Hourcade
Paul N
PN
Barlow
John P
JP
Atkinson
D001665:Binding Sites; D003179:Complement C3b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015935:Complement C4b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism / Q000469:parasitology; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052940:Merozoites / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010963:Plasmodium falciparum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017463:Receptors, Complement 3b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053838:Scattering, Small Angle; D020349:Surface Plasmon Resonance; D014961:X-Ray Diffraction
C509991:CR1 protein, human; D008565:Membrane Proteins; D015800:Protozoan Proteins; C464927:RH4 protein, Plasmodium falciparum; D017463:Receptors, Complement 3b; D003179:Complement C3b; D015935:Complement C4b
Cell Surface Receptor; Complement System; Convertases; Malaria; NMR; Plasmodium; Protein Structure; Reticulocyte-binding Homologue Proteins; Surface Plasmon Resonance (SPR)