The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II

Abstract

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.

Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

“…Last, abnormal phi values (i.e., a rare result when the ratio ΔΔ G ‡ /ΔΔ G N‐U upon mutation of an amino acid residue does not fall between zero and one) have been implicated in frustrated regions of well‐studied protein models such as chymotrypsin inhibitor 2 (CI2) or the SH3 domain 9,13 . Although observations of memory effects, severe kinetic trapping, abnormal phi values, and a lack of folding cooperativity have been reported in mature protease folding, their connection to un‐evolved folding landscapes has not been extensively examined until now 6,7,30,43,44 …”

“…Clearly, an important selection pressure on PS evolution is the spatiotemporal regulation of proteolysis, as uncontrolled proteolysis can lead to several diseases 47 . Modulation of protein folding landscapes is another likely evolutionary pressure on PS evolution in some proteins and will be the focus of the discussion in this article 4,7,43,44 . Last, the evolution of the zymogen sequence is of course closely connected to its mature sequence, which cannot evolve in such a way that prevents PS folding and must allow for enzyme catalysis and its associated dynamical motions around the native energy basin 48 …”

“…In some sense, this idea is already well established when considering proteins which are obligately folded by chaperones 63 —or the abundance of intrinsically disordered proteins which defy the structure–function paradigm and exhibit energy landscapes far from smooth funnels 64,65 . To understand this apparent contradiction in proteases, folding landscapes can be viewed as “self‐terraforming,” or determined by sequences that encode folding landscapes which permit conformational rearrangements to alter their own primary sequences, which in turn reshapes their folding landscapes 2,7,8,30 . Importantly, natural selection for exploring diverse energy landscapes does not appear to be a narrow outcome of protease folding: a variety of new studies have identified protein folding landscapes in nature which are not funnelled to the native ensemble or even to a single ensemble (Figure 2).…”

“…Pepsin and αLP remain the best‐characterized examples of PS folding, although the role of the PS during folding has been confirmed for several proteases across the major catalytic types 16 . This behavior has been reported in nearly 40 proteases, and it is likely more widespread than currently known based on conservation of PS motifs involved in the folding process 2,7,17–23 . It is important to clarify, however, that not all proteases require a PS to fold properly 8 .…”

Activation mechanism of plasmepsins, pepsin‐like aspartic proteases from Plasmodium, follows a unique trans‐activation pathway

“…Recently, we have shown that the truncated prosegment assists in correct folding of pro‐tPMII, with a mechanism similar to that of pepsin [16]. Crystal structures of pro‐tPMs have been determined for P. falciparum PMII (pro‐tPMII) [17,18], HAP (pro‐tHAP) [19], PMIV (pro‐tPMIV) [18], and P. vivax PM ( Pv ‐pro‐tPM) [20].…”