SummaryThe malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress.

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress. malaria | egress | electron tomography | soft X-ray microscopy | electron energy loss spectroscopy T he major cause of severe human malaria is Plasmodium falciparum, and its asexual blood cycle is the source of all clinical disease (1). Egress is an important step in the blood life cycle, as it allows daughter merozoites produced by intracellular parasite replication to escape and invade new erythrocytes, thereby continuing and amplifying the infection. Merozoites develop within a parasitophorous vacuole (PV), a membrane-bound compartment that forms during invasion (2-4), so the daughter parasites have two compartments to escape (5, 6).Blood-stage malaria parasites replicate by schizogony, in which several rounds of nuclear division form a multinucleated syncytium called a schizont. Individual merozoites are then produced by an unusual form of cytokinesis called budding or segmentation, which involves invagination of the single plasma membrane of the schizont. Minutes before egress, the segmented schizont suddenly transforms from an irregular to a relatively symmetrical structure with the merozoites arranged around the central digestive vacuole (5). This process, referred to as "flower formation" or rounding up, is usually accompanied by noticeable swelling of the PV and apparent shrinkage of the host cell (4, 5, 7-9). The first membrane to rupture at egress is the parasitophorous vacuole membrane (PVM) (5,6,8). When the PV does not occupy the entire infected cell, the individual merozoites can be seen to be expelled into the blood cell cytosol seconds before they escape fr...

Summary Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method’s success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.

Key Points• Plasmodium falciparumgenerated cytoadherent knobs on infected erythrocytes contain a spiral framework linked to the red cell cytoskeleton.• The findings suggest a structural basis for transmission of shear forces in adhesion of infected cells.Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells. (Blood. 2016;127(3):343-351) IntroductionPlasmodium falciparum malaria remains one of the leading causes of child deaths globally, with the majority of cases occurring in subSaharan Africa and Southeast Asia. Although chemopreventive and vector control initiatives led to an estimated 42% reduction in mortality rates between 2000 and 2012, the emergence of artemisinin resistance highlights the importance of continued efforts to understand and interfere with the biology of the parasite. 1Of the 5 Plasmodium species capable of infecting humans, P falciparum and P vivax are the most prevalent, with P falciparum causing 90% of malaria-related deaths. P falciparum-infected erythrocytes become cytoadherent, causing erythrocyte sequestration in the microvasculature and avoiding clearance of infected cells by the spleen.2 Much of the virulence of P falciparum malaria has been attributed to this cytoadherence, which impedes blood circulation and results in severe syndromes such as cerebral or placental malaria. 2-4The dominant ligand mediating cytoadherence is PfEMP1, a major variable erythrocyte...

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.

Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.

Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0Å and 2.8Å, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues.Crystallisable glycosylation mutants open up new possibilities for co-crystallisation studies to aid the design of novel ACE inhibitors. Keywordsangiotensin-converting enzyme; X-ray crystal structure; testis ACE; normal mode; hinge-bending Since its isolation in 1956 as "hypertensin-converting enzyme", human angiotensinconverting enzyme (ACE 1 ) has been known to play a key role in the regulation of blood † This work was supported by the Carnegie Corporation of New York, the University of Cape Town, the South Afican National Research Foundation and the Wellcome Trust (UK) grants 070060 and 071047. # The atomic coordinates and structure factors for glycosylation mutants tACE-G13 (codes 2iul and r2iulsf) and tACE-G1234 (codes 2iux and r2iuxsf) have been deposited in the RCSB Protein Data Bank, www.pdb.org. 1 Abbreviations: ACE, angiotensin converting enzyme 1; sACE, somatic ACE; tACE, testis ACE; NBDNJ, N-butyldeoxynojirimycin; Ndom, N-terminal domain of sACE; ACE2, human angiotensin-converting enzyme 2; NMA, normal mode analysis; tACE-G13, mutant of human tACE lacking the N-terminal 36 residues and all but the first and third N-glycosylation sites; tACE-G1234, mutant of human tACE lacking the N-terminal 36 residues and all but the first four N-glycosylation sites; ACE2o, open unliganded structure of ACE2; ACE2c, closed liganded structure of ACE2; tACEo, model of human tACE based on ACE2o; tACEc, model of human tACE based on ACE2c; NAG, N-acetyl glucosamine.

Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynD(pum)) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD(pum) mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.