Jürgen Bosch

Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP–aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-Å and 2.7-Å structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix–loop–helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.

Fragment-based cocktail crystallography by the medical structural genomics of pathogenic protozoa consortium.

The history of fragment-based drug discovery, with an emphasis on crystallographic methods, is sketched, illuminating various contributions, including our own, which preceded the industrial development of the method. Subsequently, the creation of the BMSC fragment cocktails library is described. The BMSC collection currently comprises 68 cocktails of 10 compounds that are shape-wise diverse. The utility of these cocktails for initiating lead discovery in structure-based drug design has been explored by soaking numerous protein crystals obtained by our MSGPP (Medical Structural Genomics of Pathogenic Protozoa) consortium. Details of the fragment selection and cocktail design procedures, as well as examples of the successes obtained are given. The BMSC Fragment Cocktail recipes are available free of charge and are in use in over 20 academic labs.

Crystal structure of glyceraldehyde‐3‐phosphate dehydrogenase from Plasmodium falciparum at 2.25 Å resolution reveals intriguing extra electron density in the active site

The crystal structure of D‐glyceraldehyde‐3‐phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 Å resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD+ is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD+. This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials. Proteins 2006.

Crystal structure of GAP50, the anchor of the invasion machinery in the inner membrane complex of Plasmodium falciparum

The glideosome associated protein GAP50 is an essential protein in apicomplexan parasites such as Plasmodium, Toxoplasma and Cryptosporidium, several species of which are important human pathogens. The 44.6kDa protein is part of a multi-protein complex known as the invasion machinery or glideosome, which is required for cell invasion and substrate gliding motility empowered by an actin-myosin motor. GAP50 is anchored through its C-terminal transmembrane helix into the inner membrane complex and interacts via a short six residue C-terminal tail with other proteins of the invasion machinery in the pellicle of the parasite. In this paper we describe the 1.7Å resolution crystal structure of the soluble GAP50 domain from the malaria parasite Plasmodium falciparum. The structure shows an αßßα fold with overall similarity to purple acid phosphatases with, however, little homology regarding the nature of the residues in the active site region of the latter enzyme. While purple acid phosphatases contain a phosphate bridged binuclear Fe-site coordinated by seven side chains with the Fe-ions 3.2Å apart, GAP50 in our crystals contains two cobalt ions each with one protein ligand and a distance between the Co(2+) ions of 18Å.

Structure of a ribulose 5‐phosphate 3‐epimerase from Plasmodium falciparum

The crystal structure of Pfal009167AAA, a putative ribulose 5‐phosphate 3‐epimerase (PfalRPE) from Plasmodium falciparum, has been determined to 2 Å resolution. RPE represents an exciting potential drug target for developing antimalarials because it is involved in the shikimate and the pentose phosphate pathways. The structure is a classic TIM‐barrel fold. A coordinated Zn ion and a bound sulfate ion in the active site of the enzyme allow for a greater understanding of the mechanism of action of this enzyme. This structure is solved in the framework of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. Proteins 2006.