Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress

ABSTRACT Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells. IMPORTANCE Malaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.


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Page 2 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8POL 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: X-RAY DIFFRACTION The reported resolution of this entry is 3.09 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic.The table shows the number of entries on which the scores are based.The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density.The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively.A grey segment represents the fraction of residues that are not modelled.

Metric
The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density.The numeric value is given above the bar.

Entry composition i ○
There are 3 unique types of molecules in this entry.The entry contains 7556 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Subtilisin-like protease 1. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry.The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and electron density.Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.
• Molecule 1: Subtilisin-like protease 1 Chain A: • Molecule 1: Subtilisin-like protease 1 Chain B: Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 3.54% of the height of the origin peak.No significant pseudotranslation is detected.

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There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes.The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms).The all-atom clashscore for this structure is 6.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All ( 85) close contacts within the same asymmetric unit are listed below, sorted by their clash

RNA i ○
There are no RNA molecules in this entry.
5.4 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligand geometry i ○
Of 13 ligands modelled in this entry, 8 are monoatomic -leaving 5 for Mogul analysis.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary.The Link column lists molecule types, if any, to which the group is linked.The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).There are no bond angle outliers.

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There are no chirality outliers.
There are no torsion outliers.
There are no ring outliers.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution.The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue.The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.

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• Molecule 2 is CALCIUM ION (three-letter code: CA) (formula: Ca).F