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Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1.

ABSTRACT: Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29(3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.

SUBMITTER: Tamir S

PROVIDER: S-EPMC3986192 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1.

Tamir Sagi S Rotem-Bamberger Shahar S Katz Chen C Morcos Faruck F Hailey Kendra L KL Zuris John A JA Wang Charles C Conlan Andrea R AR Lipper Colin H CH Paddock Mark L ML Mittler Ron R Onuchic José N JN Jennings Patricia A PA Friedler Assaf A Nechushtai Rachel R

Proceedings of the National Academy of Sciences of the United States of America 20140325 14

Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 a ...[more]

PMID: 24706857

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Project description:BackgroundIdentifying protein interfaces can inform how proteins interact with their binding partners, uncover the regulatory mechanisms that control biological functions and guide the development of novel therapeutic agents. A variety of computational approaches have been developed for predicting a protein's interfacial residues from its known sequence and structure. Methods using the known three-dimensional structures of proteins can be template-based or template-free. Template-based methods have limited success in predicting interfaces when homologues with known complex structures are not available to use as templates. The prediction performance of template-free methods that only rely only upon proteins' intrinsic properties is limited by the amount of biologically relevant features that can be included in an interface prediction model.ResultsWe describe the development of an integrated method for protein interface prediction (ISPIP) to explore the hypothesis that the efficacy of a computational prediction method of protein binding sites can be enhanced by using a combination of methods that rely on orthogonal structure-based properties of a query protein, combining and balancing both template-free and template-based features. ISPIP is a method that integrates these approaches through simple linear or logistic regression models and more complex decision tree models. On a diverse test set of 156 query proteins, ISPIP outperforms each of its individual classifiers in identifying protein binding interfaces.ConclusionsThe integrated method captures the best performance of individual classifiers and delivers an improved interface prediction. The method is robust and performs well even when one of the individual classifiers performs poorly on a particular query protein. This work demonstrates that integrating orthogonal methods that depend on different structural properties of proteins performs better at interface prediction than any individual classifier alone.

Project description:An active segment of the research community designing small molecules ("minimalist mimics" of peptide fragments) to interfere with protein-protein interactions have based their studies on an implicit hypothesis. Here we refer to this as the Secondary Structure Hypothesis, that might be defined as, "If a small molecule can orient amino acid side-chains in directions that resemble side-chains of the parent secondary structure at the interface, then that small molecule is a candidate to perturb the protein-protein interaction". Rigorous tests of this hypothesis require co-crystallization of minimalist mimics with protein receptors, and comparison of the bound conformations with the interface secondary structures they were designed to resemble. Unfortunately, to the best of our knowledge, there is no such analysis in the literature, and it is unlikely that enough examples will emerge in the near future to test the hypothesis. Research described here was designed to challenge this hypothesis from a different perspective. In a previous study, preferred conformations of a series of novel minimalist mimics were simulated then systematically overlaid on >240?000 crystallographically characterized protein-protein interfaces. Select data from that overlay procedure revealed chemotypes that overlay side chains on various PPI interfaces with a relatively high frequency of occurrence. The first aim of this work was to determine if good secondary structure mimics overlay frequently on PPI interfaces. The second aim of this work was to determine if overlays of preferred conformers at interface regions involve secondary structures. Thus situations where these conformations overlaid extremely well on PPI interfaces were analyzed to determine if secondary structures featured the PPI regions where these molecules overlaid in the previous study. Combining conclusions from these two studies enabled us to formulate a hypothesis that is complementary to the Secondary Structure Hypothesis, but, unlike this, is supported by abundant data. We call this the Interface Mimicry Hypothesis.

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Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1.

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Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1.

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