An Insulin-Responsive Sensor in the SIRT1 Disordered Region Binds DBC1 and PACS-2 to Control Enzyme Activity.

Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

TGF-ß2 uses the concave surface of its extended finger region to bind betaglycan's ZP domain via three residues specific to TGF-ß and inhibin-α.

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-ß family that selectively binds transforming growth factor-ß (TGF-ß) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-ß2 with BG's C-terminal binding domain, BGZP-C, and surface plasmon resonance binding measurements with TGF-ß2 variants, we found that the BGZP-C-binding site on TGF-ß2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-ß isoforms and the InhA α-subunit, but they are unconserved in other TGF-ß family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-ß2 variants with the corresponding residues from BMP-2 bound BGZP-C more weakly than corresponding alanine variants. The BGZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-ß2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-ß2 and InhA bind BGZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BGZP-C-binding site on TGF-ß2 and shed light on the specificity of BG for select TGF-ß-type GFs and the mechanisms by which BG influences their signaling.

Binding Properties of the Transforming Growth Factor-ß Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling.

Transforming growth factor (TGF) ß1, ß2, and ß3 (TGF-ß1-TGF-ß3, respectively) are small secreted signaling proteins that each signal through the TGF-ß type I and type II receptors (TßRI and TßRII, respectively). However, TGF-ß2, which is well-known to bind TßRII several hundred-fold more weakly than TGF-ß1 and TGF-ß3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-ß2 at independent sites, but how it binds TGF-ß2 to potentiate TßRII binding and how the complex with TGF-ß, TßRII, and betaglycan undergoes the transition to the signaling complex with TGF-ß, TßRII, and TßRI are not understood. To investigate the mechanism, the binding of the TGF-ßs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TßRI and TßRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-ß homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TßRII to bind. These studies further show that betaglycan modestly potentiates the binding of TßRII and must be displaced to allow TßRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-ß2 on the cell surface and thus promote the binding of TßRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TßRI, which simultaneously displaces betaglycan and stabilizes the bound TßRII by direct receptor-receptor contact.

Inhibitory protein-protein interactions of the SIRT1 deacetylase are choreographed by post-translational modification.

Regulation of SIRT1 activity is vital to energy homeostasis and plays important roles in many diseases. We previously showed that insulin triggers the epigenetic regulator DBC1 to prime SIRT1 for repression by the multifunctional trafficking protein PACS-2. Here, we show that liver DBC1/PACS-2 regulates the diurnal inhibition of SIRT1, which is critically important for insulin-dependent switch in fuel metabolism from fat to glucose oxidation. We present the x-ray structure of the DBC1 S1-like domain that binds SIRT1 and an NMR characterization of how the SIRT1 N-terminal region engages DBC1. This interaction is inhibited by acetylation of K112 of DBC1 and stimulated by the insulin-dependent phosphorylation of human SIRT1 at S162 and S172, catalyzed sequentially by CK2 and GSK3, resulting in the PACS-2-dependent inhibition of nuclear SIRT1 enzymatic activity and translocation of the deacetylase in the cytoplasm. Finally, we discuss how defects in the DBC1/PACS-2-controlled SIRT1 inhibitory pathway are associated with disease, including obesity and non-alcoholic fatty liver disease.

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro.

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

APOBEC2 is a monomer in solution: implications for APOBEC3G models.

Although the physiological role of APOBEC2 is still largely unknown, a crystal structure of a truncated variant of this protein was determined several years ago [Prochnow, C. (2007) Nature445, 447-451]. This APOBEC2 structure had considerable impact in the HIV field because it was considered a good model for the structure of APOBEC3G, an important HIV restriction factor that abrogates HIV infectivity in the absence of the viral accessory protein Vif. The quaternary structure and the arrangement of the monomers of APOBEC2 in the crystal were taken as being representative for APOBEC3G and exploited in explaining its enzymatic and anti-HIV activity. Here we show, unambiguously, that in contrast to the findings for the crystal, APOBEC2 is monomeric in solution. The nuclear magnetic resonance solution structure of full-length APOBEC2 reveals that the N-terminal tail that was removed for crystallization resides close to strand ß2, the dimer interface in the crystal structure, and shields this region of the protein from engaging in intermolecular contacts. In addition, the presence of the N-terminal region drastically alters the aggregation propensity of APOBEC2, rendering the full-length protein highly soluble and not prone to precipitation. In summary, our results cast doubt on all previous structure-function predictions for APOBEC3G that were based on the crystal structure of APOBEC2.

Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin.

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-ß family, potentiate the signaling activity of TGF-ß2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BGO) reveals an insertion that blocks the region that the endoglin orphan domain (ENGO) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-ß and BGO, as well as small-angle X-ray scattering data, BGO is shown to bind its cognate GF in an entirely different manner compared with ENGO. The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-ß family GFs.

Crystal structure and interaction studies of the human FBxo3 ApaG domain.

UNLABELLED: Transcriptional activation of proinflammatory cytokines, mediated by tumor necrosis factor receptor-associated factors (TRAFs), is in part triggered by the degradation of the F-box protein, FBxl2, via an E3 ligase that contains another F-box protein, FBxo3. The ApaG domain of FBxo3 is required for the interaction with and degradation of FBxl2 [Mallampalli RK et al., (2013) J Immunol 191, 5247-5255]. Here, we report the X-ray structure of the human FBxo3 ApaG domain, residues 278-407, at 2.0 Å resolution. Like bacterial ApaG proteins, this domain is characterized by a classic Immunoglobin/Fibronectin III-type fold, comprising a seven-stranded ß-sheet core, surrounded by four extended loops. Although cation binding had been proposed for bacterial ApaG proteins, no interactions with Mg(2+) or Co(2+) were detected for the human ApaG domain. In addition, dinucleotide polyphosphates, which have been reported to be second messengers in the inflammation response and targets of the bacterial apaG-containing operon, are not bound by the human ApaG domain. In the context of the full-length protein, loop 1, comprising residues 294-303, is critical for the interaction with FBxl2. However, titration of the individual ApaG domain with a 15-mer FBxl2 peptide that was phosphorylated on the crucial T404, as well as the inability of the ApaG domain to interact with full-length FBxl2, assessed by coimmunoprecipitation, indicate that the ApaG domain alone is necessary, but not sufficient for binding and degradation of FBxl2. DATABASE: PDB ID (5HDW).

Getting in sync with dimeric Eg5. Initiation and regulation of the processive run.

Eg5/KSP is the kinesin-related motor protein that generates the major plus-end directed force for mitotic spindle assembly and dynamics. Recent work using a dimeric form of Eg5 has found it to be a processive motor; however, its mechanochemical cycle is different from that of conventional Kinesin-1. Dimeric Eg5 appears to undergo a conformational change shortly after collision with the microtubule that primes the motor for its characteristically short processive runs. To better understand this conformational change as well as head-head communication during processive stepping, equilibrium and transient kinetic approaches have been used. By contrast to the mechanism of Kinesin-1, microtubule association triggers ADP release from both motor domains of Eg5. One motor domain releases ADP rapidly, whereas ADP release from the other occurs after a slow conformational change at approximately 1 s(-1). Therefore, dimeric Eg5 begins its processive run with both motor domains associated with the microtubule and in the nucleotide-free state. During processive stepping however, ATP binding and potentially ATP hydrolysis signals rearward head advancement 16 nm forward to the next microtubule-binding site. This alternating cycle of processive stepping is proposed to terminate after a few steps because the head-head communication does not sufficiently control the timing to prevent both motor domains from entering the ADP-bound state simultaneously.

Dimeric Eg5 maintains processivity through alternating-site catalysis with rate-limiting ATP hydrolysis.

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acid-quench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P(i) release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.

Pathway of ATP hydrolysis by monomeric kinesin Eg5.

Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k(cat), indicative that it is the rate-limiting step of the mechanism.

A structural model for monastrol inhibition of dimeric kinesin Eg5.

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.