Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.
All data generated or analysed during this study are included in the manuscript and supporting files. Key plasmids will be made available via Addgene.
- Linton M Traub
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Suzanne R Pfeffer, Stanford University School of Medicine, United States
© 2019, Traub
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.
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.
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.