Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding
- Department of Biochemistry, Brandeis University, United States
- Department of Biology, Massachusetts Institute of Technology, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- Department of Chemistry and Chemical Biology, Northeastern University, United States
Figures
Figure 1 with 2 supplements
Interaction with the N-terminus of ALAS is necessary and sufficient for activation of ALAS by an unfoldase.
(A) Peptide array of the ALAS sequence (58–548, sliding window of fifteen amino acids, shifted two amino acids towards the C-terminus with each spot, N- to C-terminus arrayed left-to-right, …
Figure 1—figure supplement 1
mtClpX-binding peptides of ALAS.
(A) Test of background antibody binding to peptide array of Δ57-ALAS sequence, as described in Figure 1A, omitting incubation with mtClpXE206Q-3xFLAG. Numbers indicate the first amino acid of the …
Figure 1—figure supplement 2
PLP occupancy of ALAS.
(A) Pyridoxal phosphate and derivatives. Modifications of the pyridoxal aldehyde group caused by conjugation in an enzyme active site or by reaction with hydroxylamine are indicated in red. (B) …
Figure 2 with 1 supplement
mtClpX relies on an unstructured N-terminal extension for rapid activation of ALAS.
(A) Edman degradation of ALAS identified a single N-terminus corresponding to amino acid 35 of the preprotein. A C-terminal Myc-His7 tag was integrated at the genomic locus encoding ALAS and the …
Figure 2—figure supplement 1
Determination and characterization of the mature N-terminus of ALAS.
(A) Western blot (mouse anti-Myc) of ALAS-Myc-His7 purified from yeast cell extracts by Ni-NTA affinity. Lanes are samples from three sequential elutions. The large arrow at right indicates major …
Figure 3 with 2 supplements
Multiple sequence-specific contacts direct mtClpX action on ALAS.
(A). The position of mutations that perturb mtClpX binding are mapped on one face of the structure of ALAS (PDB: 5TXR [Brown et al., 2018]) (F71 in yellow, Y73 in orange, and Y274 in dark blue). PLP …
Figure 3—figure supplement 1
mtClpX-binding sequences in ALAS.
15-amino-acid peptides derived from mtClpX-binding sequences determined in Figure 1A were scanned at each position with alanine and aspartate in a peptide array. The top row of the array contains …
Figure 3—figure supplement 2
Structure and function of mtClpX-interacting residues in ALAS.
(A) Spontaneous binding rates of PLP to indicated ALAS variants. Rates were determined by linear fits to the pseudolinear early phase of PLP binding, measured by fluorescence (ex. 434 nm, em. 515 …
Figure 4 with 1 supplement
mtClpX remodels a limited region of ALAS that extends from its N-terminal binding site to the active site of ALAS.
(A) Left panel: mtClpX-induced deuterium uptake in PLP-depleted (hydroxylamine-treated) ALAS (difference in deuterium uptake of ALAS with mtClpX and ATP present and ALAS with ATP only). A set of …
Figure 4—figure supplement 1
Deuterium uptake difference maps.
(A) Legend for deuterium uptake difference maps in (B–D). Legend indicates minimum value of induced deuterium uptake for each color. (B–D) Difference maps comparing the states indicated composed of …
Figure 5 with 1 supplement
Unfolding is arrested near the N-terminus of the ordered structure of ALAS.
(A) Deuterium uptake plots of peptides in ALAS (amino acid coordinates indicated) indicate a break in the linear path of ClpX-induced uptale. The most N-terminal alpha helix displays ClpX-induced …
Figure 5—figure supplement 1
KM for ATP hydrolysis by E. coli ClpX.
Reactions were performed in a 384-well plate at 30°C in PD150 with 0.3 μM ClpX hexamer, indicated concentrations of ATP, and an NADH-oxidation-coupled assay system (1 mM NADH, 7.5 mM …
Figure 6 with 1 supplement
Unfolding of N-terminal secondary structure is required for mtClpX to activate ALAS.
(A) The proposed path of mtClpX unfolding of ALAS to the active site is indicated in blue on the structure of yeast ALAS (PDB: 5TXR (Brown et al., 2018); image created with UCSF Chimera (Pettersen …
Figure 6—figure supplement 1
Model for mtClpX-directed remodeling and activation of ALAS.
A group of sequence-specific contacts (blue sidechains) position mtClpX on ALAS; this contacts are important for both avidity and efficiency of processing. A flexible N-terminal element extending …
Additional files
-
Supplementary file 1
Deuterium uptake and difference values for all peptides monitored in ALAS.
- https://cdn.elifesciences.org/articles/54387/elife-54387-supp1-v2.xlsx
-
Supplementary file 2
S. cerevisiae strains used in this work.
All strains were made in w303 mat a background (MATa ade2-1 leu2-3 ura3 trp1-1 his3-11,15 can1-100 GAL psi+).
- https://cdn.elifesciences.org/articles/54387/elife-54387-supp2-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/54387/elife-54387-transrepform-v2.pdf
