Biology subjects

Purhonen, P.

Publications and source records attributed to Purhonen, P..

3 recordsLinked to original sources

bioRxiv2023

The membrane-cytoplasmic linker defines activity of FtsH proteases

Pandemic Pseudomonas aeruginosa clone C strains encode a xenolog of FtsH (PaFtsH2), an inner-membrane associated ATP-dependent protease. FtsH1 supports growth and intrinsic antibiotic resistance but cannot be replaced by ftsH2. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Swapping residues of a short MC peptide that links transmembrane helix-2 with the cytosolic AAA+ ATPase module from PaFtsH1 into PaFtsH2 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. FtsH1 MC peptides are glycine rich. Introducing three glycines into the membrane-proximal end of PaFtsH2s MC linker is sufficient to elevate activity in vitro and in vivo. Electron microscopy including PaFtsH2 indicates that MC linker identity influences FtsH flexibility. Our findings establish that the efficiency of substrate processing by two PaFtsH isoforms depends on how they are attached to the membrane and suggest that greater linker flexibility/length allows FtsH to degrade a wider spectrum of substrates. As FtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is not a latent enzyme, rather recognizes specific substrates or is activated in specific contexts or biological niches. We hypothesize that such linkers might play a more determinative role in functionality and physiological impact of FtsH proteases than previously thought.

biochemistry↗

bioRxiv2020

CHD7 interacts with the nucleosome acidic patch for its efficient activity via its N-terminal region

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed integrative biophysical analysis on CHD7 chromatin remodeler using crosslinking-mass spectrometry (XL-MS), cryo-Electron Microscopy (cryo-EM) and single-molecule Forster Resonance Energy Transfer (smFRET). We uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. The cryo-EM analysis on the N-CRD_Chromodomain bound to nucleosome reveals that the N-CRD interacts with the acidic patch of nucleosome. Furthermore, smFRET analysis shows the mutations in the N-CRD result in slow or highly-fluctuating remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

biochemistry↗

bioRxiv2020

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

biochemistry↗