Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex Michael M. Kariuki a,1 , Xuerong Li a,1 , Innocent Yamodo a , Athar H. Chishti b , Steven S. Oh a, * a Division of Cell Biology, Caritas St. ElizabethÕs Medical Center, Tufts University School of Medicine, Boston, MA, USA b Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, USA Received 28 September 2005 Available online 2 November 2005 Abstract Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein 1 and 9 (MSP1, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19 kDa C-terminal domain of MSP1 (MSP1 19 ). Two specific regions of MSP9 defined as D1a and D2 interacted with native MSP1 19 . The 42 kDa domain of MSP1 (MSP1 42 ) bearing MSP1 19 in the C-terminus bound directly to both MSP9/D1a and D2. Thus, the regions of MSP1 and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSP1, MSP9, and band 3 during erythrocyte invasion by P. falciparum. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Plasmodium falciparum; Malaria; Merozoite surface protein; Erythrocyte; Band 3; Invasion; Co-ligand complex; Protein interaction The invasion of erythrocytes by Plasmodium falciparum is a multi-step process requiring specific interactions between the merozoite and host erythrocyte proteins [1]. Earlier studies have suggested that P. falciparum may favor the sialic acid-dependent or sialic acid-independent mecha- nism to invade erythrocytes [2–5]. In the sialic acid-depen- dent invasion pathway, two micronemal proteins, the 175 kDa erythrocyte binding antigen (EBA-175) and the 140 kDa erythrocyte-binding antigen (EBA-140; also known as EBP-2 and BAEBL) have been identified as par- asite ligands binding to erythrocyte sialoglycoprotein receptors, glycophorin A [6–8] and glycophorin C [9–12], respectively. A 5 0 cysteine-rich motif known as the Duffy binding-like domain of EBA-175 and EBA-140 is thought to be involved in the binding of the erythrocyte receptor [8,9]. Furthermore, P. falciparum normocyte-binding pro- tein 1 (PfNBP-1, also known as P. falciparum reticulo- cyte-binding homologue protein 1) interacts with an unidentified trypsin-resistant erythrocyte receptor Y in a sialic acid-dependent manner [13]. A recent study has shown that PfNBP-1 is required for sialic acid-dependent invasion of erythrocytes by P. falciparum [14]. Receptor E which is a hypothetical erythrocyte sialoglycoprotein resistant to trypsin and sensitive to chymotrypsin has been proposed to interact with P. falciparum JESEBL (also known as EBA-181) by a sialic acid-dependent mechanism [15]. In the sialic acid-independent invasion pathway, recep- tor X defined as a trypsin-sensitive erythrocyte receptor has been proposed to interact with an unidentified P. falci- parum merozoite protein [2,16,17]. P. falciparum reticulo- cyte-binding homologue protein 2b (PfRh2b; also known as PfNBP-2b) is thought to interact with the 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.154 * Corresponding author. Fax: +1 617 789 3111. E-mail address: steven.oh@tufts.edu (S.S. Oh). 1 These authors contributed equally to this work and are listed alphabetically. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 338 (2005) 1690–1695 BBRC