Mandar T. Naik

Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation.

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response

While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.

Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain α-Ketoacid Dehydrogenase Complex

The purified mammalian branched-chain α-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain α-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other α-ketoacid dehydrogenase complexes.

Quality carbon fibers from fractionated lignin

The application of lignin carbon fibers was hindered by their low quality and mechanical performance. We addressed this challenge by developing a new approach to fractionate and modify lignin to produce quality carbon fibers using an enzyme-mediator system, which derives lignin fractions with different molecular weights, functional groups, and interunitary linkages. The fractionated lignin in general improves the miscibility and spinnability of lignin. In particular, the insoluble lignin fraction renders carbon fibers with a significantly improved turbostratic carbon structure as revealed by XRD and Raman spectroscopy. The improvement in the carbon structure leads to the significantly improved elastic modulus. The results suggest that higher molecular weights, less –OH groups, and more linear structures may contribute to the improved crystallization and mechanical performance of lignin carbon fibers. The technical breakthrough produces lignin-based carbon fibers with a similar elastic modulus to commercial carbon fibers for the first time, and paves the path for replacing PAN with lignin for producing quality carbon fibers.

A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L.

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encoding a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.

Molecular weight and uniformity define the mechanical performance of lignin-based carbon fiber

Lignin-based carbon fiber represents a renewable and low-cost alternative to petroleum-based carbon fiber. However, the poor mechanical performance of current lignin carbon fiber hinders its application. We hypothesized that the lower optimal mechanical performance is caused by the inherent heterogeneity of lignin. It is still unknown how the molecular weights (MWs) and lignin uniformity will impact the performance of lignin carbon fiber. We thereby addressed this hypothesis by fractionating lignin into fractions with different MWs and polydispersity indices (PDIs). An enzyme–mediator-based method and a dialysis method were developed to derive lignin fractions with increased MW and decreased PDI. Lignin fractions were electro-spun into fibers after blending with polyacrylonitrile (PAN) at 1 : 1 (w/w) ratio. The fractionation in general improved the spinnability of lignin to allow us to obtain finer lignin-based carbon fibers. The elastic modulus of lignin carbon fibers, as measured by nanoindentation, was increased as the lignin MW increased and as PDI decreased. The scatter plot and linear regression revealed very good correlation between the elastic modulus and PDI, as well as certain correlation between the elastic modulus and MW. XRD and Raman spectroscopy revealed that the crystallite size and the content of the pre-graphitic turbostratic carbon increased with higher lignin MW and lower PDI, revealing the mechanism of the improvement in carbon fiber mechanical performance. This study elucidated the impacts of lignin MW and uniformity on the mechanical properties of carbon fiber, and could thus guide the development of lignin processing technologies for quality lignin-based carbon fiber.

Structural analysis of poly-SUMO chain recognition by the RNF4-SIMs domain

The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain α‐ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain α‐ketoacids. Recently hbLBD has been found to follow two‐step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small β‐barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two‐step; native state ↔denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 ± 0.9 K. Cold denaturation of hbLBD is also predicted and discussed.

Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase complex

A reversible two‐step (native state↔denatured state) folding mechanism based on equilibrium and stopped flow experiments is proposed for urea denaturation of the lipoyl‐bearing domain (hbLBD) of human mitochondrial branched chain α‐ketoacid dehydrogenase (BCKD) complex. The results from this circular dichroism (CD) and fluorescence study have ruled out populated kinetic or equilibrium intermediates on folding pathway of this β‐barrel domain under experimental conditions. Both studies suggested mono‐exponential kinetics without any burst phases. Moreover the thermodynamic parameters ΔG H2O and m obtained from the kinetic analysis are consistent with the equilibrium measurements.

NMR chemical shift assignments of a complex between SUMO-1 and SIM peptide derived from the C-terminus of Daxx

Small Ubiquitin-like MOdifiers (SUMOs) are ubiquitin-like proteins known to covalently modify large number of cellular proteins. The mammalian SUMO family includes four paralogues, SUMO-1 through SUMO-4. Death-associated protein-6, Daxx, is a 740 residue important transcription corepressor known to represses transcriptional potential of several sumolyted transcription factors. Daxx also plays important role in apoptosis. Both terminals of Daxx harbor separate SUMO Interaction Motifs (SIM), which mediate its interaction with SUMO and hence the sumolyted transcription factors. The C-terminal SIM of Daxx preferentially binds SUMO-1. Practically complete 1H, 13C and 15N resonance assignments for the complex between SUMO-1 and 20 residue Daxx C-terminal SIM peptide are reported here.