Molecular determinants of phase separation for Drosophila DNA replication licensing factors
Abstract
Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.
Data availability
All data generated during this study is included in the manuscript and supporting files.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R01GM141045-01)
- James M Berger
- Michael R Botchan
National Cancer Institute (R01CA030490)
- James M Berger
- Michael R Botchan
National Institute of General Medical Sciences (F32GM116393)
- Matthew W Parker
UC Berkeley Jessie Rabinowitz Award
- Jonchee A Kao
Cancer Prevention and Research Institute of Texas (RR200070)
- Matthew W Parker
Welch Foundation (I-2074-20210327)
- Matthew W Parker
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Stephen P Bell, Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
Version history
- Preprint posted: April 12, 2021 (view preprint)
- Received: June 1, 2021
- Accepted: December 23, 2021
- Accepted Manuscript published: December 24, 2021 (version 1)
- Version of Record published: February 3, 2022 (version 2)
Copyright
© 2021, Parker 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.
Metrics
-
- 1,845
- Page views
-
- 288
- Downloads
-
- 4
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Evolutionary Biology
Evolution can tinker with multi-protein machines and replace them with simpler single-protein systems performing equivalent functions in an equally efficient manner. It is unclear how, on a molecular level, such simplification can arise. With ancestral reconstruction and biochemical analysis, we have traced the evolution of bacterial small heat shock proteins (sHsp), which help to refold proteins from aggregates using either two proteins with different functions (IbpA and IbpB) or a secondarily single sHsp that performs both functions in an equally efficient way. Secondarily single sHsp evolved from IbpA, an ancestor specialized in strong substrate binding. Evolution of an intermolecular binding site drove the alteration of substrate binding properties, as well as the formation of higher-order oligomers. Upon two mutations in the α-crystallin domain, secondarily single sHsp interacts with aggregated substrates less tightly. Paradoxically, less efficient binding positively influences the ability of sHsp to stimulate substrate refolding, since the dissociation of sHps from aggregates is required to initiate Hsp70-Hsp100-dependent substrate refolding. After the loss of a partner, IbpA took over its role in facilitating the sHsp dissociation from an aggregate by weakening the interaction with the substrate, which became beneficial for the refolding process. We show that the same two amino acids introduced in modern-day systems define whether the IbpA acts as a single sHsp or obligatorily cooperates with an IbpB partner. Our discoveries illuminate how one sequence has evolved to encode functions previously performed by two distinct proteins.
-
- Biochemistry and Chemical Biology
- Computational and Systems Biology
Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. However, the high sequence and structural conservation of the catalytic kinase domain complicates the development of selective kinase inhibitors. Inhibition of off-target kinases makes it difficult to study the mechanism of inhibitors in biological systems. Current efforts focus on the development of inhibitors with improved selectivity. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target effects. We develop a multicompound-multitarget scoring (MMS) method that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables optimization of inhibitor combinations for multiple on-targets. Using MMS with published kinase inhibitor datasets we determine potent inhibitor combinations for target kinases with better selectivity than the most selective single inhibitor and validate the predicted effect and selectivity of inhibitor combinations using in vitro and in cellulo techniques. MMS greatly enhances selectivity in rational multitargeting applications. The MMS framework is generalizable to other non-kinase biological targets where compound selectivity is a challenge and diverse compound libraries are available.