Search

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl–coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first x-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the Central and UCS domains form a contiguous arrangement of seventeen consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains. Small-angle x-ray scattering data suggest that free DmUNC-45 adopts an elongated conformation and exhibits flexibility in solution. Protease sensitivity maps to a conserved loop that contacts the most carboxy-terminal UNC-45 armadillo repeat sub-domain. Amino acid conservation across diverse UCS proteins maps to one face of this carboxy-terminal sub-domain and the majority of mutations that affect myosin-dependent cellular activities lie within or around this region. Our crystallographic, biophysical, and biochemical analyses suggest that DmUNC-45 function is afforded by its flexibility and by structural integrity of its UCS domain.

UNC-45 is a chaperone that may aid in folding myosin's motor domain. Mutations in UNC-45 cause muscle defects and dysfunction, due to the importance of myosin in muscle structure and contraction. UNC-45 is composed of an N-terminal tetra-tricopeptide (TPR) domain, a C-terminal UCS (UNC-45/Cro1/She4 homology) domain, and a central region that links these. Here we present the crystal structure of Drosophila UNC-45 (dUNC-45), which should serve as a basis for attaining a detailed understanding of its mechanism of action. Bacterially expressed recombinant His-tagged dUNC-45 was purified sequentially using immobilized metal affinity chromatography and size exclusion chromatography. The protein eluted as a single peak, indicating a homogeneous population of protein suitable for crystallization. Crystals were prepared by hanging drop vapor diffusion and x-ray diffraction data were collected to a limit of 3.0 A resolution. For phase determination, a seleno-methionine derivative dUNC-45 crystal was prepared for single wavelength anomalous dispersion (SAD) experiments. Synchrotron data were collected at the Berkeley National Laboratory Advanced Light Source. The diffraction data were processed in HKL2000. Selenium positions were determined and refined in Phenix. The resulting structure was refined against native data using Phaser and maximum-likelihood refinement with Refmac5. Model building was performed in COOT. Our current model has R-cryst and R-free values of 0.24 and 0.28 respectively. The TPR domain is not visible in the model, likely due to flexibility of the domain within the crystal. Overall, the UCS domain is composed of α-helices that form armadillo repeats which stack together to form a supercoiled structure. Based on our structure, it appears that the central and UCS domains make up a single structural unit. Currently, we are pursuing structure-based biochemical assays to pinpoint the protein surfaces that are involved in the binding and chaperone activities of dUNC-45.