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• W2000805968 abstract "The protozoan parasite Plasmodiumcauses malaria, with hundreds of millions of cases recorded annually. Protection against malaria infection can be conferred by antibodies against merozoite surface protein (MSP)-1, making it an attractive vaccine candidate. Here we present the structure of the C-terminal domains of MSP-1 (known as MSP-119) from Plasmodium knowlesi. The structure reveals two tightly packed epidermal growth factor-like domains oriented head to tail. In domain 1, the molecule displays a histidine binding site formed primarily by a highly conserved tryptophan. The protein carries a pronounced overall negative charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-119, we have analyzed the crystal contacts in five different crystal environments, revealing that domain 1 is highly preferred in protein-protein interactions. A comparison of MSP-119 structures fromP. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are similar, the molecules show significant differences in charge distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might prevent invasion of the merozoite into red blood cells. The protozoan parasite Plasmodiumcauses malaria, with hundreds of millions of cases recorded annually. Protection against malaria infection can be conferred by antibodies against merozoite surface protein (MSP)-1, making it an attractive vaccine candidate. Here we present the structure of the C-terminal domains of MSP-1 (known as MSP-119) from Plasmodium knowlesi. The structure reveals two tightly packed epidermal growth factor-like domains oriented head to tail. In domain 1, the molecule displays a histidine binding site formed primarily by a highly conserved tryptophan. The protein carries a pronounced overall negative charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-119, we have analyzed the crystal contacts in five different crystal environments, revealing that domain 1 is highly preferred in protein-protein interactions. A comparison of MSP-119 structures fromP. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are similar, the molecules show significant differences in charge distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might prevent invasion of the merozoite into red blood cells. merozoite surface protein epidermal growth factor root mean square A necessary step in the life cycle of the malaria parasite is its entry into the red blood cell of a mammalian host. This process involves many surface proteins specifically expressed at the merozoite stage of the parasite, including merozoite surface protein (MSP)1-1 (also known as merozoite surface antigen-1 or MSA-1). MSP-1, a required component of the merozoite invasion machinery, associates noncovalently with other parasite proteins, including the C-terminal portion of MSP-6 (1Trucco C. Fernandez-Reyes D. Howell S. Stafford W.H. Scott-Finnigan T.J. Grainger M. Ogun S.A. Taylor W.R. Holder A.A. Mol. Biochem. Parasitol. 2001; 112: 91-101Crossref PubMed Scopus (97) Google Scholar). MSP-1 is synthesized as a 180–225-kDa polypeptide capable of binding sialic acid (2Perkins M.E. Rocco L.J. J. Immunol. 1988; 141: 3190-3196PubMed Google Scholar, 3Su S. Sanadi A.R. Ifon E. Davidson E.A. J. Immunol. 1993; 151: 2309-2317PubMed Google Scholar). As a requirement for merozoite entry into a red cell, MSP-1 undergoes two processing steps, the first at merozoite release from an infected cell and the second during invasion of a red cell (4Holder A.A. Blackman M.J. Burghaus P.A. Chappel J.A. Ling I.T. McCallum-Deighton N. Shai S. Mem. Inst. Oswaldo Cruz. 1992; 87 Suppl. 3: 37-42Crossref PubMed Scopus (151) Google Scholar) (Fig. 1 a). In the first processing step, the precursor polypeptide is cleaved into four chains (with apparent molecular masses of 83, 30, 38, and 42 kDa) held together by noncovalent contacts (4Holder A.A. Blackman M.J. Burghaus P.A. Chappel J.A. Ling I.T. McCallum-Deighton N. Shai S. Mem. Inst. Oswaldo Cruz. 1992; 87 Suppl. 3: 37-42Crossref PubMed Scopus (151) Google Scholar, 5Holder A.A. Sandhu J.S. Hillman Y. Davey L.S. Nicholls S.C. Cooper H. Lockyer M.J. Parasitology. 1987; 94: 199-208Crossref PubMed Scopus (122) Google Scholar, 6McBride J.S. Heidrich H.G. Mol. Biochem. Parasitol. 1987; 23: 71-84Crossref PubMed Scopus (178) Google Scholar). In the second processing step, the 42-kDa segment (MSP-142) is cleaved into two fragments with apparent molecular masses of 33 kDa (MSP-133) and 19 kDa (MSP-119 or MSP-1inv) (4Holder A.A. Blackman M.J. Burghaus P.A. Chappel J.A. Ling I.T. McCallum-Deighton N. Shai S. Mem. Inst. Oswaldo Cruz. 1992; 87 Suppl. 3: 37-42Crossref PubMed Scopus (151) Google Scholar). MSP-133sheds from the surface, whereas MSP-119 remains anchored to the merozoite membrane by a glycosylphosphatidylinositol tail attached to the C-terminal residue (7Blackman M.J. Heidrich H.G. Donachie S. McBride J.S. Holder A.A. J. Exp. Med. 1990; 172: 379-382Crossref PubMed Scopus (479) Google Scholar, 8Blackman M.J. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 307-315Crossref PubMed Scopus (185) Google Scholar, 9Blackman M.J. Ling I.T. Nicholls S.C. Holder A.A. Mol. Biochem. Parasitol. 1991; 49: 29-33Crossref PubMed Scopus (234) Google Scholar, 10Blackman M.J. Whittle H. Holder A.A. Mol. Biochem. Parasitol. 1991; 49: 35-44Crossref PubMed Scopus (132) Google Scholar). Because high antibody titer against the C-terminal region of MSP-1 protects against severe malaria, this molecule represents an attractive vaccine candidate (11al-Yaman F. Genton B. Kramer K.J. Chang S.P. Hui G.S. Baisor M. Alpers M.P. Am. J. Trop. Med. Hyg. 1996; 54: 443-448Crossref PubMed Scopus (107) Google Scholar, 12Egan A.F. Morris J. Barnish G. Allen S. Greenwood B.M. Kaslow D.C. Holder A.A. Riley E.M. J. Infect. Dis. 1996; 173: 765-769Crossref PubMed Scopus (280) Google Scholar, 13Riley E.M. Allen S.J. Wheeler J.G. Blackman M.J. Bennett S. Takacs B. Schonfeld H.J. Holder A.A. Greenwood B.M. Parasite Immunol. (Oxf.). 1992; 14: 321-337Crossref PubMed Scopus (208) Google Scholar). Antibodies against MSP-119correlate with clinical immunity (14Shi Y.P. Sayed U. Qari S.H. Roberts J.M. Udhayakumar V. Oloo A.J. Hawley W.A. Kaslow D.C. Nahlen B.L. Lal A.A. Infect. Immun. 1996; 64: 2716-2723Crossref PubMed Google Scholar, 15O'Donnell R.A. de Koning-Ward T.F. Burt R.A. Bockarie M. Reeder J.C. Cowman A.F. Crabb B.S. J. Exp. Med. 2001; 193: 1403-1412Crossref PubMed Scopus (234) Google Scholar); however, recombinant MSP-119 is poorly immunogenic, presumably due to poor processing of the highly disulfide-bonded protein (16Egan A.F. Chappel J.A. Burghaus P.A. Morris J.S. McBride J.S. Holder A.A. Kaslow D.C. Riley E.M. Infect. Immun. 1995; 63: 456-466Crossref PubMed Google Scholar, 17Egan A. Waterfall M. Pinder M. Holder A. Riley E. Infect. Immun. 1997; 65: 3024-3031Crossref PubMed Google Scholar). Antibodies to the C-terminal region of MSP-1 fall into two categories: protective antibodies that inhibit merozoite invasion of red cells, and blocking antibodies that enhance invasion by interfering with the binding of protective antibodies. Protective antibodies include those that recognize the native conformation of MSP-119 (7Blackman M.J. Heidrich H.G. Donachie S. McBride J.S. Holder A.A. J. Exp. Med. 1990; 172: 379-382Crossref PubMed Scopus (479) Google Scholar, 18Chang S.P. Gibson H.L. Lee-Ng C.T. Barr P.J. Hui G.S. J. Immunol. 1992; 149: 548-555PubMed Google Scholar, 19Hui G.S. Chang S.P. Gibson H. Hashimoto A. Hashiro C. Barr P.J. Kotani S. J. Immunol. 1991; 147: 3935-3941PubMed Google Scholar, 20Locher C.P. Tam L.Q. Vaccine. 1993; 11: 1119-1123Crossref PubMed Scopus (12) Google Scholar), those that block proteolytic processing of MSP-142 (21Blackman M.J. Scott-Finnigan T.J. Shai S. Holder A.A. J. Exp. Med. 1994; 180: 389-393Crossref PubMed Scopus (226) Google Scholar), and certain antibodies to the 83-kDa N-terminal fragment of intact MSP-1 (22Guevara Patino J.A. Holder A.A. McBride J.S. Blackman M.J. J. Exp. Med. 1997; 186: 1689-1699Crossref PubMed Scopus (212) Google Scholar). Blocking antibodies bind to the first domain of MSP-119, near the N terminus introduced by the processing of MSP-142 (22Guevara Patino J.A. Holder A.A. McBride J.S. Blackman M.J. J. Exp. Med. 1997; 186: 1689-1699Crossref PubMed Scopus (212) Google Scholar, 23Chappel J.A. Egan A.F. Riley E.M. Druilhe P. Holder A.A. Infect. Immun. 1994; 62: 4488-4494Crossref PubMed Google Scholar). The extremely stable MSP-119 contains two consecutive epidermal growth factor (EGF)-like domains, a small disulfide-rich fold also found in diverse proteins including Factor Xa (24Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar), E-selectin (25Graves B.J. Crowther R.L. Chandran C. Rumberger J.M. Li S. Huang K.S. Presky D.H. Familletti P.C. Wolitzky B.A. Burns D.K. Nature. 1994; 367: 532-538Crossref PubMed Scopus (389) Google Scholar), cyclooxygenase-2 (26Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1613) Google Scholar), and integrin αVβ3 (27Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar). In MSP-1, disulfide bonds are integral to the correct folding of the molecule (20Locher C.P. Tam L.Q. Vaccine. 1993; 11: 1119-1123Crossref PubMed Scopus (12) Google Scholar). Although the full-length MSP-1 is highly polymorphic, the C-terminal EGF-like domains in MSP-119 are much less so, and they represent a conserved segment of a variable molecule. MSP-119 shows high sequence identity across Plasmodium species: P. knowlesi MSP-119 shares 82% sequence identity withP. cynomolgi and 51% sequence identity with P. falciparum. To better understand the process of invasion of thePlasmodium merozoite into a red blood cell, we have solved the structure of the C-terminal domains of MSP-1 from P. knowlesi by x-ray crystallography. We have refined the structure to an R-factor of 23.4% and R-free of 26.4% to 2.4 Å resolution (Table I). Our structure reveals features of MSP-1 novel to P. knowlesiand provides new insight into MSP-1 from otherPlasmodium species. With four copies in the crystallographic asymmetric unit, we observe MSP-1 in four different environments, allowing us to infer novel properties of the molecule.Table ICrystallographic statisticsDataSpace groupP21Cell lengths (Å)33.9, 106.5, 62.7Cell angles (°)90, 102.1, 90Resolution (Å) (last shell)50–2.4 (2.49–2.4)No. of observations (last shell)56,495 (4,653)No. of unique observations (last shell)16,570 (1,565)Completeness (%) (last shell)97.7 (93.7)R-sym (last shell)aR-sym = ΣhΣi‖I h,i-〈I h〉‖/ΣhΣi‖I h,i‖, where Ih,i is the ithintensity measurement of reflection h, and 〈Ih〉 is the average intensity of that reflection.0.082 (0.315)〈I/ς1〉 (last shell)11.6 (3.4)RefinementR-work/R-freebR-work/R-free = Σh‖FP −FC‖/Σh‖FP‖, whereFC is the calculated and FP is the observed structure factor amplitude of reflection h for the working/free set, respectively.23.4%/26.4%No. of atomsProtein2720Water306Other20RamachandranFavored85.5%Allowed14.5%Generous0%Forbidden0%r.m.s. deviationsBonds0.008 ÅAngles1.3 °Dihedrals26.1 °Impropers0.88 °a R-sym = ΣhΣi‖I h,i-〈I h〉‖/ΣhΣi‖I h,i‖, where Ih,i is the ithintensity measurement of reflection h, and 〈Ih〉 is the average intensity of that reflection.b R-work/R-free = Σh‖FP −FC‖/Σh‖FP‖, whereFC is the calculated and FP is the observed structure factor amplitude of reflection h for the working/free set, respectively. Open table in a new tab DNA encoding the C-terminal 92 amino acids of the of P. knowlesi (Malayan H strain) MSP-1 (GenBankTM accession code AAG24615) was inserted into the vector YEpRPEU-3 and expressed in the Saccharomyces cerevisiae VK1 cell line (36Stowers A.W. Keister D.B. Muratova O. Kaslow D.C. Infect. Immun. 2000; 68: 5530-5538Crossref PubMed Scopus (51) Google Scholar). The vector included an N-terminal yeast α-factor pre-pro secretory signal and a C-terminal hexahistidine tag. The plasmid is episomally maintained by supplying a functional HIS4 gene for the host VK1 cell line (his4). Protein expression is under control of the ADH2 promoter and induced by introducing ethanol as a carbon source during fermentation. The secreted protein product included 105 amino acids: 5 amino acids (E-A-E-A-S) from the cleaved mating factor, the 92 amino acids from MSP-1, and 8 amino acids (G-P-H6) from the affinity tag. The protein was purified using nickel affinity, ion exchange, and gel filtration chromatography. Purified MSP-119 protein was concentrated to 10 mg/ml for crystallization trials. Crystals were grown via vapor diffusion with a solution containing 30% polyethylene glycol 6000, 100 mmHEPES, pH 7.0. Crystals were transferred into the same solution supplemented with 20% glycerol before cooling to 100 K in a nitrogen stream. X-ray data were collected on a Rigaku RU-200 rotating anode generator equipped with nickel mirrors and a RAXIS 4 detector (Molecular Structure Corp., The Woodlands, TX). 180° of diffraction data were processed using DENZO (37Otwinowski Z. Minor W. Carter C.W. Sweet R.M. Methods in Enzymology: Macromolecular Crystallography, Part A. 276. Academic Press, New York1997: 307-326Google Scholar), SCALEPACK (37Otwinowski Z. Minor W. Carter C.W. Sweet R.M. Methods in Enzymology: Macromolecular Crystallography, Part A. 276. Academic Press, New York1997: 307-326Google Scholar), and TRUNCATE (38Collaborative Computational Project, Number 4. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19796) Google Scholar) to a resolution limit of 2.4 Å. Molecular replacement was performed with the AMoRe package (38Collaborative Computational Project, Number 4. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19796) Google Scholar) using coordinates from the C-terminal domains of MSP-1 from P. cynomolgi (29Chitarra V. Holm I. Bentley G.A. Petres S. Longacre S. Mol. Cell. 1999; 3: 457-464Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The P. cynomolgi model was rotated and translated against the 8–4 ÅP. knowlesi diffraction amplitudes, using correlation coefficients to rank solutions. Four successive objects were placed in the crystallographic asymmetric unit, whereas insertion of a fifth object failed to improve the correlation coefficient. Inspection of the packing showed no steric clashes in a unit cell with 49% solvent content. Rigid body refinement in the programs AMoRe (38Collaborative Computational Project, Number 4. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19796) Google Scholar) and Crystallography and NMR System (CNS) (39Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) was followed by model building in the program O (40Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta. Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Residue numbering of the P. knowlesi structure corresponds to the mature secreted protein and begins at the first residue of the cleaved α mating factor secretory signal. Refinement protocols in CNS included conjugate gradient minimization, simulated annealing, and temperature factor refinement. Models were built into simulated annealing composite omit maps calculated in CNS. Tight 4-fold noncrystallographic symmetry restraints (300 kcal/mol-Å2) were imposed on all atoms in the early stages of refinement and later relaxed for atoms that differ among the four copies in the asymmetric unit. Refinement steps were accepted only if they reduced the R-free (of a test set comprised of 860 reflections, 5% of the total, selected using resolution shells). TheR-work and R-free are 23.4% and 26.4%, respectively, using all reflections to 2.4 Å. Least squares superpositions were performed using the program LSQMAN (41Kleywegt G.J. Read R.J. Structure. 1997; 5: 1557-1569Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) with a distance cutoff of 3.8 Å, and coordinate transformations were applied using the program MOLEMAN2 (41Kleywegt G.J. Read R.J. Structure. 1997; 5: 1557-1569Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Structural comparisons with the NMR structure of P. falciparum MSP-119 (28Morgan W.D. Birdsall B. Frenkiel T.A. Gradwell M.G. Burghaus P.A. Syed S.E. Uthaipibull C. Holder A.A. Feeney J. J. Mol. Biol. 1999; 289: 113-122Crossref PubMed Scopus (134) Google Scholar) use the most representative member of the ensemble. Electrostatic calculations include only the native MSP-119 sequence, without the secretory signal and purification epitope. Molecular figures were prepared using the programs MOLSCRIPT (42Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOBSCRIPT (43Esnouf R.M. J. Mol. Graph. Model. 1997; 15: 132-134Crossref PubMed Scopus (1795) Google Scholar), and GRASP (44Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar). The C-terminal region ofP. knowlesi MSP-1 contains two successive EGF-like domains packed together tightly and related by a rotation of ∼170° (Fig.1 b). The molecule is roughly square-shaped, with dimensions of about 35 Å × 35 Å × 15 Å. The angle between domains 1 and 2 leads to close approach of the N terminus of domain 1 and the C terminus of domain 2. Thus, as domain 2 is anchored in the membrane by a glycosylphosphatidylinositol linkage attached to the C-terminal residue, the N-terminal residue of domain 1 (i.e. the cleavage site in MSP-142) also points toward the membrane, in an appropriate orientation for processing by proteases attached to the merozoite membrane (28Morgan W.D. Birdsall B. Frenkiel T.A. Gradwell M.G. Burghaus P.A. Syed S.E. Uthaipibull C. Holder A.A. Feeney J. J. Mol. Biol. 1999; 289: 113-122Crossref PubMed Scopus (134) Google Scholar). Each EGF-like domain in P. knowlesi MSP-1 contains a segment of random coil followed by four antiparallel β strands (Fig.1 b). Typically, EGF-like domains contain six cysteines in three disulfide bonds with a connectivity of 1–3, 2–4, and 5–6. The MSP-1 EGF-like domains follow this paradigm, except that in domain 1, valine and tryptophan replace the middle (2Perkins M.E. Rocco L.J. J. Immunol. 1988; 141: 3190-3196PubMed Google Scholar, 3Su S. Sanadi A.R. Ifon E. Davidson E.A. J. Immunol. 1993; 151: 2309-2317PubMed Google Scholar, 4Holder A.A. Blackman M.J. Burghaus P.A. Chappel J.A. Ling I.T. McCallum-Deighton N. Shai S. Mem. Inst. Oswaldo Cruz. 1992; 87 Suppl. 3: 37-42Crossref PubMed Scopus (151) Google Scholar) disulfide. The valine and tryptophan pack into the domain 1 hydrophobic core, in the same volume as the disulfide bond in canonical EGF-like domains. The crystals of P. knowlesi MSP-119 contain four molecules in the asymmetric unit, revealing the protein in four different environments. The four molecules are highly similar, with r.m.s. deviations of 0.4–0.6 Å for 77–81 Cα atoms, with differences primarily at the two termini and residues 69–74 on the loop between β strands 1 and 2 in domain 2 (Fig. 1 c). This loop shows differences across the four molecules in the crystallographic asymmetric unit and also within one molecule (as reflected by high B factors). The flexibility in this loop and in the termini corresponds to the mobility seen in the NMR structure of theP. falciparum MSP-119 (28Morgan W.D. Birdsall B. Frenkiel T.A. Gradwell M.G. Burghaus P.A. Syed S.E. Uthaipibull C. Holder A.A. Feeney J. J. Mol. Biol. 1999; 289: 113-122Crossref PubMed Scopus (134) Google Scholar). In P. knowlesi MSP-119, the extensive interface between the two domains buries 26 residues and 1090 Å2 of surface area. This produces a rigid interface with little variation in the interdomain angle among the four copies in the asymmetric unit (Fig. 1 c). The residues in the interface are among the most conserved in the MSP-119 protein: 23 of 26 residues are identical between P. knowlesi and P. cynomolgi, with three conservative substitutions; and 14 of 26 residues are identical between P. knowlesi and P. falciparum, with seven conservative substitutions. The highly conserved domain 2 sequence Asn57-Asn58-Gly59-Gly60packs in the interface between domains 1 and 2, forming a short stretch of left-handed helix in P. knowlesi and P. cynomolgi and a tight turn in P. falciparum. P. knowlesi domain 1 superimposes on domain 2 with an r.m.s. deviation of 1.2–1.6 Å for the four independent copies in the asymmetric unit (Table II).Table IIComparison of MSP-119 structuresMSP-119 fromP. knowlesi (4 copies)MoleculeABCDResidues in model8–988–1009–1018–94Mean B factor49.451.557.862.3No. of residues in crystal contacts37373121D1–D2 angle168°168°174°173°D1–D2 r.m.s.d.ar.m.s.d., r.m.s. deviation; PDB, Protein Data Bank.1.6 Å (23 Cα atoms)1.4 Å (23 Cα atoms)1.2 Å (23 Cα atoms)1.3 Å (24 Cα atoms)Contents of His binding siteHis B100His C101His from DHis from AMSP-119 structures across speciesP. knowlesiP. cynomolgiP. falciparumPDB code1N1I1B9W1CEJNo. of residues928996Sequence ID vs. P. knowlesi100%82%51%Overall charge−7−4−2r.m.s.d. vs. P. knowlesicopy A0.4–0.6 Å (77–81 Cα atoms)1.3 Å (78 Cα atoms)1.7 Å (65 Cα atoms)a r.m.s.d., r.m.s. deviation; PDB, Protein Data Bank. Open table in a new tab In all four copies of MSP-119 in the crystallographic asymmetric unit, electron density appears in a shallow depression on the surface (Fig.2 a). In each copy, the density traces back to a histidine from the hexahistidine tag of a symmetry-related molecule. For each of the four bound histidines, the plane of the imidazole ring stacks roughly parallel to the plane of the indole ring of Trp34, and the carboxylic acid of Glu42 pairs with the bound (and presumably protonated) histidine. In the histidine-binding pocket, Trp34 forms the floor, and Glu33 and Arg35 form the walls (Fig.2 b). The hydrophobic tryptophan and the bound histidine appear in the middle of a region of negative electrostatic potential (Fig. 2 c). Although the conformation of the C-terminal hexahistidine tail differs in the four copies in the asymmetric unit, one histidine from the tail fills a histidine-binding pocket of a symmetry-related molecule. In the four copies in the asymmetric unit, each histidine binding buries between 167 and 206 Å2 of accessible surface area. This same pocket can bind other positively charged moieties: in the crystal of MSP-119 from P. cynomolgi (29Chitarra V. Holm I. Bentley G.A. Petres S. Longacre S. Mol. Cell. 1999; 3: 457-464Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), a lysine from a symmetry-related molecule inserts into this pocket. In all Plasmodium species except P. falciparum, domain 1 of MSP-119 lacks the middle disulfide bond found in canonical EGF-like domains. The replacement of a disulfide by a small residue (valine, isoleucine, or threonine) and a tryptophan produces a shelf on domain 1 capable of binding histidine. Tryptophan is the residue most commonly found buried in protein-protein interfaces (30Glaser F. Steinberg D.M. Vakser I.A. Ben-Tal N. Proteins. 2001; 43: 89-102Crossref PubMed Scopus (344) Google Scholar) because burial of its large hydrophobic surface area yields thermodynamic gain. The analysis of crystal contacts can be used to map protein binding surfaces on a molecule to reveal potential biologically important surfaces on the molecule (31Crosio M.P. Janin J. Jullien M. J. Mol. Biol. 1992; 228: 243-251Crossref PubMed Scopus (75) Google Scholar, 32Garman S.C. Sechi S. Kinet J.P. Jardetzky T.S. J. Mol. Biol. 2001; 311: 1049-1062Crossref PubMed Scopus (46) Google Scholar). We searched crystal contacts in MSP-119 to identify the favored sites for binding to proteins. Combining the four copies ofP. knowlesi MSP-119 in the crystallographic asymmetric unit with the one copy of P. cynomolgiMSP-119 (29Chitarra V. Holm I. Bentley G.A. Petres S. Longacre S. Mol. Cell. 1999; 3: 457-464Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), we have observations of MSP-119in five different crystal environments. We counted the number of times each residue participates in crystal contacts and mapped that number onto the surface of the protein (Fig.3 a). The P. knowlesi and P. cynomolgi crystal contacts correlate: of the 9 residues involved in crystal contacts in all four P. knowlesi copies, 7 make contacts in the P. cynomolgicrystals as well. The seven residues (Ile14, Asp15, Leu28, Trp34, Glu42, Ala50, and Ser51) participate in crystal contacts in all five crystal packing environments. All of these except Ser51 are conserved between P. knowlesi and P. cynomolgi, suggesting protein binding sites conserved across species. All of these except Leu28 differ between P. knowlesi andP. falciparum, resulting in distinctly shaped MSP-119 surfaces across these species. The crystal contacts show that MSP-119 domain 1 is highly favored for binding protein (Fig. 3 a). Trp34, the floor of the histidine binding site in the center of domain 1, falls in an ideal position to participate in protein-protein interactions on the surface of the merozoite. In P. falciparum MSP-119, domain 1 has been shown to be the binding site of monoclonal antibody G17.12 (33Pizarro J.C. Chitarra V. Calvet C. Verger D. Bentley G.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1246-1248Crossref PubMed Scopus (9) Google Scholar). The high level of sequence identity in MSP-119 across Plasmodium species results in highly similar three-dimensional structures of the molecule (Figs.3 b and 4 a). TheP. cynomolgi MSP-119 superimposes on the P. knowlesi structure with an r.m.s. deviation of 1.3 Å for 78 Cα atoms, whereas the P. falciparum structure superimposes on the P. knowlesi structure with an r.m.s. deviation of 1.7 Å for 65 Cα atoms (Table II). A superposition of the four P. knowlesi MSP-119molecules with the P. cynomolgi and P. falciparumhomologues reveals that the differences occur primarily at the termini and in the 69–74 loop, the same loop that shows variability among the four molecules in the P. knowlesi crystal. MSP-119 from P. knowlesi exhibits a striking charge distribution (Fig. 4 b). The molecule carries a pronounced negative potential, with an overall charge of −7 in the 92 residues of the two domains. There are no buried ion pairs in P. knowlesi MSP-119; rather, all charged residues are surface-accessible. The overall charge is species-specific because the equivalent segments of MSP-1 from P. cynomolgi and P. falciparum have overall charges of −4 and −2, respectively (Fig.4, c and d). In P. knowlesiMSP-119, the negative charge is concentrated in domain 2 (with an overall charge of −7), whereas domain 1 is neutrally charged overall. The acidic regions in domain 1 cluster near the histidine binding site. Hydrophobicity maps of the three PlasmodiumMSP-119 structures show a patch of nonpolar residues along the interface between the two domains (Fig. 4, b−d). Of the 26 residues contributing to the interface, 15 are hydrophobic, and 3 are cysteines in disulfide bonds. Because the molecule is only 15 Å thick, some hydrophobic residues in the interdomain interface (including Tyr25, Tyr27, Leu37, Leu38, and Phe88) remain partially solvent-accessible. Surprisingly, the large patch of hydrophobic residues on the back surface is among the least active surfaces in protein binding, as judged by the crystal contact analysis (Fig.3 a). The arrangement of charged and hydrophobic residues on MSP-119 may maintain specificity of the interactions between MSP-119 and its binding partners such as the other MSP-1 fragments and MSP-6. Complementary changes between residues in MSP-119 and its binding partners may allow evolution of the MSP-119 protein surface. The region around Trp34 appears to be a site onP. knowlesi MSP-119 well suited for protein-protein interactions. First, all EGF-like domain structures contain at least three pairs of disulfide bonds, with one exception: the first domain of MSP-119. Of the 40 independent EGF/laminin-like domain structures currently in the Protein Data Bank (34Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27887) Google Scholar), only Plasmodium MSP-119 has lost one of its three canonical disulfide bonds. The loss of a structurally important and highly conserved disulfide bond is most likely due to a functional requirement. Second, all of thePlasmodium species (except P. falciparum) contain a tryptophan in domain 1 of MSP-119 in place of the canonical cysteine. The disulfide is replaced with a tryptophan and a smaller residue; whereas the smaller residue may become a valine, isoleucine, or threonine, the other member of the pair is always tryptophan, suggesting functional selection. Third, tryptophan is the residue most commonly found in protein-protein interfaces (30Glaser F. Steinberg D.M. Vakser I.A. Ben-Tal N. Proteins. 2001; 43: 89-102Crossref PubMed Scopus (344) Google Scholar), so the replacement of a strictly conserved disulfide-forming cysteine with a tryptophan suggests the evolution of a protein binding site. Fourth, there are no instances of crystals of MSP-119 with an empty histidine-binding pocket. In all five independent crystallographic environments, a histidine or lysine is found stacked above the tryptophan ring. This pocket fills when the concentration of nearby protein becomes sufficiently high. Fifth, the crystal contact map of MSP-119 indicates that the tryptophan and nearby regions in domain 1 are very active in participating in protein-protein interactions. The sequence differences between P. falciparum and the otherPlasmodium species represent divergent evolution from a common precursor, but it is unclear whether P. falciparumhas lost a binding site or the other Plasmodium species have evolved new functionality. Alternatively, the difference betweenP. falciparum and the other Plasmodia may be due to the strong bias against tryptophan in P. falciparum (35Tchavtchitch M. Fischer K. Huestis R. Saul A. Mol. Biochem. Parasitol. 2001; 118: 211-222Crossref PubMed Scopus (26) Google Scholar). The virulent P. falciparum displays somewhat different disease pathology compared with the other Plasmodiumspecies; perhaps the differences in MSP-119 proteins are related to the differences in disease severity. For example, P. falciparum invades a broader spectrum of red blood cells and reaches higher levels of parasitemia compared with other malaria species, potentially due to loss of receptor specificity in P. falciparum. Some of this specificity may be conferred by the MSP-1 protein during red blood cell invasion. Although P. falciparum lacks the tryptophan defining the floor of the binding pocket, all the other species of Plasmodium have this tryptophan, which generates a site for binding histidine. We propose the use of this histidine binding site as a target for the design of compounds that bind, for example, P. vivax MSP-1. From such a scaffold compound, new inhibitors that protect against merozoite invasion of a red blood cell might be developed. Our structure reveals a new feature of the MSP-119molecule, a histidine-binding pocket in domain 1. Because antibodies that protect against malarial infections often bind specifically to domain 1, this new property of the molecule might be used to engineer tighter binding and thus more protective antibodies. Alternatively, this histidine-binding pocket might be used to interfere with the binding of blocking antibodies. For example, a small molecule inhibitor containing an imidazole scaffold may be designed to overlap with the footprint of blocking antibodies and thus disrupt the binding of blocking antibodies. The specific disruption of MSP-119blocking antibodies without perturbation of protective antibodies would greatly enhance the immune response against the malaria parasite. We thank T. Allison and the other members of the Structural Biology Section for discussions." @default.
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• W2000805968 title "Structure of the C-terminal Domains of Merozoite Surface Protein-1 from Plasmodium knowlesi Reveals a Novel Histidine Binding Site" @default.
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