ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1.
- Johanna Ludwigsen
- Ashish K Singh
- Martin Zacharias
- Felix Mueller-Planitz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Jerry L Workman, Stowers Institute for Medical Research, United States
© 2017, Ludwigsen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Downloads (link to download the article as PDF)
Download citations (links to download the citations from this article in formats compatible with various reference manager tools)
Open citations (links to open the citations from this article in various online reference manager services)
The >800 human G protein–coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.
The infective ability of the opportunistic pathogen Staphylococcus aureus, recognized as the most frequent cause of biofilm-associated infections, is associated with biofilm mediated resistance to host immune response. Phenol-soluble modulins (PSM) comprise the structural scaffold of S. aureus biofilms through self-assembly into functional amyloids, but the role of individual PSMs during biofilm formation remains poorly understood and the molecular pathways of PSM self-assembly have yet to be identified. Here, we demonstrate high degree of cooperation between individual PSMs during functional amyloid formation. PSMα3 initiates the aggregation, forming unstable aggregates capable of seeding other PSMs resulting in stable amyloid structures. Using chemical kinetics we dissect the molecular mechanism of aggregation of individual PSMs showing that PSMα1, PSMα3 and PSMβ1 display secondary nucleation whereas PSMβ2 aggregates through primary nucleation and elongation. Our findings suggest that the various PSMs have solved to ensure fast and efficient biofilm formation through cooperation between individual peptides.