Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy.

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs. SIGNIFICANCE: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.

Engineering Allostery into Proteins.

Our ability to engineer protein structure and function has grown dramatically over recent years. Perhaps the next level in protein design is to develop proteins whose function can be regulated in response to various stimuli, including ligand binding, pH changes, and light. Endeavors toward these goals have tested and expanded on our understanding of protein function and allosteric regulation. In this chapter, we provide examples from different methods for developing new allosterically regulated proteins. These methods range from whole insertion of regulatory domains into new host proteins, to covalent attachment of photoswitches to generate light-responsive proteins, and to targeted changes to specific amino acid residues, especially to residues identified to be important for relaying allosteric information across the protein framework. Many of the examples we discuss have already found practical use in medical and biotechnology applications.

Energy and Enzyme Activity Landscapes of Yeast Chorismate Mutase at Cellular Concentrations of Allosteric Effectors.

In solution, proteins fluctuate among many conformational substates, with their relative free energies determining substate populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the homodimeric Saccharomyces cerevisiae chorismate mutase (ScCM) enzyme, which is negatively and positively regulated by tyrosine and tryptophan, respectively. Previous X-ray crystallography structures revealed two equivalent allosteric binding pockets that can be occupied by either tryptophan or tyrosine. We proposed that under cellular conditions there are six potential states of ScCM: no allosteric effector bound, a single tyrosine bound, a single tryptophan bound, two tyrosines bound, two tryptophans bound, and a mixed bound state in which tyrosine and tryptophan occupy different allosteric sites. We used isothermal titration calorimetry and solution-state nuclear magnetic resonance spectroscopy to confirm the existence of all six states and construct the complete six-state equilibrium binding profile. We were also able to assign enzyme activities to each state, which allowed us to derive the enzyme activity landscape across the range of cellular concentrations of tyrosine and tryptophan. Surprisingly, the mixed bound state had the highest enzyme activity, which suggested that the shikimate pathway is shunted toward tyrosine production under most conditions.

Assigning methyl resonances for protein solution-state NMR studies.

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.

Biophysical and computational methods to analyze amino acid interaction networks in proteins.

Globular proteins are held together by interacting networks of amino acid residues. A number of different structural and computational methods have been developed to interrogate these amino acid networks. In this review, we describe some of these methods, including analyses of X-ray crystallographic data and structures, computer simulations, NMR data, and covariation among protein sequences, and indicate the critical insights that such methods provide into protein function. This information can be leveraged towards the design of new allosteric drugs, and the engineering of new protein function and protein regulation strategies.