Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

a highly fluorescent N-terminal product. Calculated of the intact substrate and

. Primary sequence of P. falciparum SERA5 and positions of proteolytic processing sites.
Shown is the complete primary sequence of the P. falciparum 3D7 SERA5 gene product (PlasmoDB ID: PF3D7_0207600). Positions of cleavage by PfSUB1 are indicated in red (site 2 and site 3, as well as the allele-specific site 1; (Yeoh et al., 2007, Debrabant et al., 1992, Li et al., 2002). The position of cleavage by protease X (Leu842), defined in this study as the P50C cleavage site, is also indicated.
The sequence encompassed by the terminal P50 processing fragment is shaded in pink and the papain-like domain Ser558-Lys832 is underlined, with the predicted catalytic triad residues Ser596, His762 and Asn787 shaded in yellow and marked with asterisks. The N-terminal secretory signal peptide is shaded in grey. Purified recombinant proteins rSERA5-C and rSERA5-A (in 25 mM HEPES pH 7.4, 150 mM NaCl) were incubated either alone or as a mixture containing approximately equivalent amounts (~2 g) of the purified proteins, and sampled either immediately (start) or after 4 h at 37 o C for analysis by SDS PAGE and Coomassie staining. The rSERA5-C underwent extensive degradation over the period of the incubation (compare the 1 st and 4 th tracks from the left), whereas rSERA5-A was essentially stable (compare the 3 rd and 6 th tracks from the left). The SERA5-A also remained stable in the mixed samples (compare the 2 nd and 5 th tracks), indicating that the rSERA5-C preparation does not contain a protease that is capable of degrading rSERA5-A in trans. Note that the batches of both proteins used for this experiment displayed more degradation immediately following purification than the (different) batches used for the experiment presented in Fig. 2B of the main manuscript. A. Reversed-phase (RP)-HPLC elution chromatograms of mock-digested and rSERA5-C-digested TACE substrate IV (Abz-LAQAVRSSSR-Dpa). Digestions were carried out for 4 h at 37 o C in 100 l 25 mM Bis-Tris buffer pH 6.5 containing 1 mM EDTA, 10 mM DTT and 0.05% (w/v) Tween 20, using a substrate concentration of 10 M and either no protein additions (mock digested) or 1 g purified rSERA5-C. Digests were then fractionated by RP-HPLC chromatography on a Vydac 4.6 mm  25 cm C 18 reversed-phase column, eluted at 1 ml min -1 with a 18-54% (v/v) gradient of acetonitrile in 0.1% TFA over 15 min, with continuous monitoring of eluted components using a Jasco FP-920 fluorescence detector ( ex 320 nm,  em 420 nm). RP-HPLC-analysis of undigested substrate, or substrate incubated under similar conditions with purified rSERA5wt or rSERA5-A, produced elution profiles identical to that of the mock-digested substrate in the top panel (not shown). The intact substrate eluted as a weakly fluorescent peak at 11.2 min, whilst in the fully digested samples a major fluorescent peak appeared at 7.8 min. Identities of the indicated peaks, including the predicted and measured masses of the major digestion product as determined by electrospray mass spectrometry, are shown. The minor fluorescent digestion product eluting at 7.02 min could not be identified, but presumably is a result of less frequent cleavage at another position in the substrate peptide backbone.
B. Schematic structure of Abz-LAQAVRSSSR-Dpa (Jin et al., 2002). In the intact peptide the fluorescence of the N-terminal donor Abz group is quenched by the accepting Dpa group. Cleavage at any point in the peptide backbone produces a highly fluorescent N-terminal product. Calculated molecular masses of the intact substrate and the Abz-LAQAVR major cleavage product obtained in this study are indicated. (1.25 M) in 100 l 25 mM Bis-Tris buffer pH 6.5 containing 1 mM EDTA, 10 mM DTT and 0.05% (w/v) Tween 20, was supplemented with 1 g purified rSERA5-C and incubated at 21 o C. Increase in fluorescence was continuously monitored with time (at 5 min intervals) as described previously (Blackman et al., 2002) using a Cary Eclipse fluorescence spectrophotometer (Varian) equipped with a 96-well microplate reader accessory ( ex 320 nm,  em 420 nm). The curve displayed represents individual reads from a single well of a kinetic assay; readings taken from triplicate wells in the same experiments showed no more than 4% variation from the plot shown at individual time-points.
B. Plot of initial hydrolysis rate (V) against substrate concentration. Initial rates were calculated under conditions in which <10% of substrate had been hydrolysed, based on progress curves such as that shown in (A).
C. Lineweaver-Burk transformation of the data shown in (B). The line of best fit was calculated by linear regression in Microsoft Excel. The intersect of the plot with the y-axis provides a measure of V max (the hydrolysis rate under conditions of substrate saturation, calculated as 8.8 RFU min -1 ), whilst the slope of the plot equates to K m /V max . The K m value (the substrate concentration at which the hydrolysis rate is equal to half of V max ) is calculated in this experiment as 11.3 M.  ORF produced following genomic integration of targeting constructs.
A. RT-PCR was performed using total RNA purified from 3D7 parental P. falciparum, or various drugresistant parasite clones obtained following transfection of the constructs described in Fig. 3A of the main manuscript. Primer pairs used (indicated) were designed to detect either mRNA transcribed from the chimeric SERA5 locus resulting from the integration event depicted in Fig. 3A (primers +27 plus -11), or an mRNA transcript similar to that from the endogenous gene, which could potentially arise by extended splicing of run-through transcript of the modified locus (primers +27 plus -25).
Parasite clones used as a source of RNA were: clone i (transfected with pHH1SERA5chimWT but displaying no integration of this construct; see Fig. S5B above); clone h (chimWT); and clone d (chimS596A). The results showed that primers +27 plus -25 produced an RT-PCR product only from parasites harbouring an unmodified SERA5 locus, indicating that the unlikely possibility of runthrough transcription and splicing to produce a wild-type mature SERA5 mRNA transcript did not take place in parasites harbouring the chimeric SERA5 locus. As expected, primers +27 plus -11 amplified a product only from the clones that had integrated the pHH1-based constructs. PCR reactions performed without reverse transcriptase (RT) usually gave no product. Where trace product is evident, this is likely due to the fact that the RNA used in these experiments was not DNase-treated before the RT reaction. However, because of the presence of intron sequence the products derived from these trace amounts of genomic DNA template migrate at a higher molecular weight than the mRNA-derived products. M, molecular mass marker DNA ladder (SmartLadder, Eurogentec). The primer pairs used for RT-PCR, and the predicted sizes of the resulting amplicons  Depicted are views facing the 'active site cleft' of the P. falciparum SERA5 papain-like domain (PDB ID: 2WBF) represented as a green molecular surface, with Ser596 shown in red. The wild-type protein is depicted on the left-hand side, whilst the predicted structure resulting from substitution of Ser596 with Arg is shown on the right. The much larger Arg side chain (shown as blue sticks with its surface as a blue mesh) is predicted to extend into the cleft and interfere with binding of putative client molecules. The figures were prepared with PyMol (http://pymol.org/).  Assay buffer conditions: a 25 mM HEPES pH 6.5, 12.5 mM CHAPS, 10 mM CaCl 2 ; b 25 mM HEPES pH 6.5, 1 mM DTT; c 25 mM HEPES pH 6.5, 1 mM DTT, 20 mM CHAPS, 1.5 mM CaCl 2 ; d 50 mM HEPES pH 6.5, 25 mM CHAPS, 10 mM CaCl 2 , 1 mM DTT; e 25 mM HEPES pH 7.4, 25 mM CHAPS, 12 mM CaCl 2 ; f 25 mM HEPES pH 7.4, 15 mM CaCl 2 , 2 mM DTT; g 25 mM Bis-Tris pH 6.5, 1 mM EDTA, 5 mM DTT; h 50 mM sodium acetate pH 5.5, 5 mM MgCl 2 , 5 mM DTT. i C(Rho), terminal Cys residues labelled with 6-iodoacetamido tetramethylrhodamine, produced as described previously (Blackman et al., 2002, Koussis et al., 2009, Yeoh et al., 2007, Withers-Martinez et al., 2012. j Peptide hydrolysis was assessed by fractionation of digestion reactions on a Vydac 4.6 mm  25 cm C 18 reversed-phase column, eluted at 1 ml min -1 with a 0-55% (v/v) gradient of acetonitrile in 0.1% (v/v) TFA over 60 min. k Fluorogenic peptide hydrolysis was monitored continuously at room temperature as previously described (Blackman et al., 2002) using a Varian Cary Eclipse fluorescence spectrophotometer by measuring the increase in fluorescence of AMC or Abz using the indicated excitation and emission wavelengths.