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Molecular basis of substrate recognition and degradation by human presequence protease - PubMed

  • ️Wed Jan 01 2014

Molecular basis of substrate recognition and degradation by human presequence protease

John V King et al. Structure. 2014.

Abstract

Human presequence protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing an ∼13,300-Å(3) catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and β-amyloid (Aβ), the latter of which contributes to Alzheimer disease pathogenesis. Here, we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and have their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP.

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Figures

Figure 1
Figure 1. Conserved Mechanisms for Substrate Capture by hPreP

(A) M16 metalloprotease domain organization. (B) Crystal structure of hPreP (C) Volumetric analysis of the hPreP catalytic chamber, performed with 3V (Voss and Gerstein, 2010). (D) Electrostatic surface representations of hPreP, atPreP (PDB: 2FGE) and Fln (PDB: 3S5H) catalytic chambers at ±6 kT/e, performed with APBS (Baker et al., 2001). Positive surfaces are blue, negative ones red, and neutral ones white. (E) Analysis of M16C catalytic chamber conservation, performed with ConSur (Ashkenazy et al., 2010). Positions are colored on a sliding scale from magenta (most conserved) to teal (degenerate). See also Figure S1 and S2.

Figure 2
Figure 2. Residues in the hPreP Chamber Facilitate Substrate Recognition

(A) HPreP-N catalytic chamber. (B) Two pockets (L111, F123, F124, and L127; and H896 and R888) explain observed cleavage site preference for P1 or P′1 hydrophobic residues, and scissile bonds 2-5 residues distal from substrate C-termini. (C) Labeled acidic residues are proximal to the active site and can facilitate interaction with basic substrate residues (D) A network of hydrophobic resides in hPreP-N permits capture of hydrophobic residues.

Figure 3
Figure 3. Role of the Linker in Catalysis

(A) L557E and P558G sit in a conserved hydrophobic pocket on the D1 surface. The lower inset is colored by electrostatic surface; the upper one, by conservation. (B) Specific activities of hPreP WT and mutants at an enzyme concentration of 1.25 nM. Activity was determined by monitoring the cleavage of 0.5μM Substrate V at 37 °C. (C) WT and mutant hPreP specific activities at indicated Substrate V concentrations. Datasets were fit to the Michaelis-Menten equation, with Vmax (98, 29, 43 s−1) and Km (26, 82, 26 μM), calculated for WT, L557E, and P558G, respectively. Mean ± SD represents at least 3 experiments. See also Figure S3.

Figure 4
Figure 4. Mass Analysis of Aβ-degradation by hPreP

(A) MALDI-ToF/ToF and (B) deconvoluted Q-ToF mass spectra of Aβ40 alone (lower panel) and hPreP-degraded Aβ40 (upper panels). Aβ40 and hPreP were mixed at the indicated molar ratios (1:100-1000). (C) Schematic of hPreP's Aβ40 cleavage sites. Filled arrows denote sites identified by (Falkevall et al., 2006), while dashed arrows indicate cleavage sites identified in this study. Asterisks mark cleavage sites reported by (Chow et al., 2009). See also Figure S4.

Figure 5
Figure 5. Structural Basis of Aβ Capture and Recognition by HPreP

(A) Aβ-binding to hPreP-N. (B) Hydrophobic exosite. Four Aβ residues (x1, y1-3) are observed. Hydrophobic residues that comprise the putative side chain binding pockets are displayed in gray. (C) Aβ-binding to the hPreP catalytic cleft. The S1 site consists of L111, F123, F124, and R900. (D-E) 2Fo-Fc electron density maps contoured at 0.5σ for Aβ residues bound to (D) the catalytic cleft and (E) the hydrophobic exosite. Arrows denote preferred Aβ cleavage sites by hPreP. See also Figure S5.

Figure 6
Figure 6. Conformational Profiles of hPreP in Solution

Pair distribution functions and scattering curves for WT (A and B), E107Q (C and D), and E107Q + Aβ (E and F). Curve fitting was performed based on atomic models input to CRYSOL (single models) and OLIGOMER (multiple models). The volume fractions shown below the profiles indicate the percent composition by conformational state in solution that yielded the line of best fit for the observed data (mixture). See also Figure S6.

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