A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A

Abstract

Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

Figure 1. Sequence of pro-cd-InpA

Sequence of the pro-domain (Ala1-Asn111) and the catalytic domain (Val112-Pro359) of pro-interpain A with interstrain differences (our unpublished results; vertical red arrows) and alignment with S. pyogenes pro-SpeB (pro-domain: Asp1P_SPE-Lys118P_SPE; mature protease domain: Gln1_SPE-Pro253_SPE; numbering according to PDB entry 1dki; (19)). Identical residues are displayed over orange background (28% sequence identity). Amino-acid residues not present in the respective 3D structures are depicted in blue. The four autolytic cleavage points of pro-cd-InpA are indicated by blue scissors. The main cleavage points of either protein leading to the stable mature forms are characterised by blue scissors and light green background. The presently studied protein, ΔN1pro-cd-InpA C154A, includes all the residues from Ala39 onwards and a predicted N-terminal helix in the N-terminal missing region is shown in pink.

Figure 2. Expression and activity of InpA

(A) Expression and purification of pro-cd-InpA C154A. Lanes 1 and 2, E. coli homogenate before and 3h after protein expression induction, respectively. Lane 3, recombinant protein after affinity chromatography purification. Molecular masses of the distinct protein species (40 and 27 kDa) are shown on the left. (B) Same for wt pro-cd-InpA. (C) Time-course analysis of autocatalytic processing and activation of wt pro-cd-InpA (final concentration 10µM) incubated with 1mM HgCl₂. The reaction was initiated by adding EDTA (5mM final concentration) as a Hg²⁺-chelator, i.e. by releasing metal-mediated inhibition. Samples were withdrawn at the time intervals specified (O/N, overnight incubation; lane C, pro-cd-InpA alone). (D) Same as (C) but after addition of active InpA (10nM final concentration) to the reaction mixture. In this case, the reaction proceeded much faster. (E) A subset volume of the withdrawn aliquots from (C) and (D) was used to quantify the activity released from wt pro-cd-InpA in the absence (○) and presence (●) of catalytic amounts of wt cd-InpA. As a control, pro-cd-InpA spiked with InpA but without EDTA was incubated in parallel (▼). (F) Concentration dependent autoactivation of pro-cd-InpA. The reaction was initiated by releasing Hg²⁺-mediated inhibition in mixtures containing 0.04µM (●), 2µM (○) and 10µM (▲) zymogen. At indicated time points, 50µl (●), 10µl (○), and 2µl (▲) were withdrawn from each reaction mixture and directly assayed for activity. (G) SDS-PAGE of the digestion of pro-cd-InpA C154A by wt cd-InpA. The zymogen (final concentration of 10µM) was incubated with cd-InpA (0.1µM) for time intervals as specified (lane C, control pro-cd-InpA C154 incubated alone). N-terminal amino acid sequences of pro-cd-InpA derived fragments are indicated on the right. (H) Western blot analysis of culture supernatant of P. intermedia using InpA-specific rabbit antiserum (lane 3). Wt cd-InpA and pro-cd-InpA (C154A) were loaded on lane 1 and 2, respectively, for comparison.

Figure 3. Experimental electron density maps

(A) Representative example of the F_obs-type map obtained after multistep-density modification, contoured at 1σ above threshold and superimposed with the model placed in accordance to the Patterson search calculations (black stick model; residues 145–148, see PDB 1pvj) and the final refined co-ordinates of ΔN1pro-cd-InpA C154A (light-grey stick model; residues 115–123). (B) (mF_obs-DF_calc)-type σ_A-weighted omit map contoured above +1.75σ above threshold illustrating the distinct chain trace around the zymogenic hairpin of molecule A (final model in light-grey) as compared with the ΔN1pro-cd-InpA model employed for phasing (black stick model). Some residues of either structure are labelled in the respective grey-tone for reference. (C) Same as (B) but showing the latency flap.

Figure 4. Structures of ΔN1pro-cd-InpA C154A and wt cd-InpA

(A) Richardson diagram of ΔN1pro-cd-InpA C154A in standard orientation. The pro-domain is displayed in blue/cyan and the mature protein moiety (subdivided into a right and a subdomain) in yellow/brown. The subdomains, the regular secondary structure elements (see Fig. 1), the N- and the C-terminus, the primary activation point (at Asn111-Val112) and the structure regions responsible for latency maintenance are marked and labelled. (B) Superimposition of the Cα-carbon traces of ΔN1pro-cd-InpA C154A (yellow) and wt mature cd-InpA (red) in standard orientation. Some residues of ΔN1pro-cd-InpA C154A are labelled for reference. (C) Close-up view of the active site of ΔN1pro-cd-InpA C154A. Orientation as in (B) after a horizontal rotation of ~45°. (D) Same as in (C) but for wt active cd-InpA. (E) Cα-trace of the structure of ΔN1pro-cd-InpA C154A (yellow) and wt mature cd-InpA (red) around the active site including ①, the catalytic cysteine residue (Cys154; mutated to alanine in ΔN1pro-cd-InpA C154A), imbedded in active-site helix α2; ②, the zymogenic hairpin encompassing the catalytic histidine (His305) (undefined from Ser295 to Gln301 in ΔN1pro-cd-InpA C154A); ③, the backing helix α1 (absent in cd-InpA); ④, the latency-flap, displayed from Tyr332 to Met351 for either structure. The grey arrows indicate the displacements of the keynote structural elements upon zymogen activation as explained in the text.

Figure 5. Comparison of ΔN1pro-cd-InpA C154A with pro-SpeB

(A) Superimposition of the Cα-carbon traces of ΔN1pro-cd-InpA C154A (blue) and pro-SpeB (yellow) in standard orientation revealing the large-scale structural deviations of the activation segments. Some residues of ΔN1pro-cd-InpA C154A are labelled for reference. (B) Detail of the region around the active site including segments shown under Fig. 4e, i.e. ①, the catalytic cysteine residue (Cys154; alanine in ΔN1pro-cd-InpA C154A), within active-site helix α2, depicted for its residues Gly153-Ala158 (pro-cd-InpA numbering, see Fig. 1); ②, the zymogenic hairpin including the catalytic histidine (His305), shown for Tyr291-Phe307; ③, the backing helix α1 from the pro-domain, shown for Pro87-Ala95; ④, the segment containing the latency-flap, displayed from Gly340 to Asp350.