30 research outputs found
Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.
The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved Ļ-x-x-Ļ-Ļ motifs (with Ļ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2APBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2AAD1(1-37) complex indicated that E2A-AD1 adopts an Ī±-helical structure and uses its Ļ-x-x-Ļ-Ļ motif to bind TAZ2. While this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2
The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300
Abstract The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma
Headgroup-Dependent Membrane Catalysis of ApelināReceptor Interactions Is Likely
Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelināAPJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-<i>sn</i>-glycero-3-[phospho-<i>rac</i>-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6āK12 is highly structured, with R6āL9 directly interacting with headgroups of the micelle. Type I Ī²-turns are initiated between R6 and L9, and a well-defined type IV Ī²-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptideāreceptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced
Preserved Transmembrane Segment Topology, Structure, and Dynamics in Disparate Micellar Environments
Detergent micelles
are frequently employed as membrane mimetics
for solution-state membrane protein nuclear magnetic resonance spectroscopy.
Here we compare topology, structure, psāns time-scale dynamics,
and hydrodynamics of a model protein with one transmembrane (TM) segment
(residues 1ā55 of the apelin receptor, APJ, a G-protein-coupled
receptor) in three distinct, commonly used micellar environments.
In each environment, two solvent-protected helical segments connected
by a solvent-exposed kink were observed. The break in helical character
at the kink was maintained in a helix-stabilizing fluorinated alcohol
environment, implying that this structural feature is inherent. Molecular
dynamics simulations also substantiate favorable self-assembly of
compact proteināmicelle complexes with a more dynamic, solvent-exposed
kink. Despite the observed similarity in TM segment behavior, micelle-dependent
differences were clear in the structure, dynamics, and compactness
of the 30-residue, extramembrane N-terminal tail of the protein. This
would affect intermolecular interactions and, correspondingly, the
functional state of the membrane protein
Conserved structural features anchor biofilm-associated RTX-adhesins to the outer membrane of bacteria
\u3cp\u3eRepeats-in-toxin (RTX) adhesins are present in many Gram-negative bacteria to facilitate biofilm formation. Previously, we reported that the 1.5-MDa RTX adhesin (MpIBP) from the Antarctic bacterium, Marinomonas primoryensis, is tethered to the bacterial cell surface via its N-terminal Region I (RI). Here, we show the detailed structural features of RI. It has an N-terminal periplasmic retention domain (RIN), a central domain (RIM) that can insert into the Ī²-barrel of an outer-membrane pore protein during MpIBP secretion, and three extracellular domains at its C terminus (RIC) that transition the protein into the extender region (RII). RIN has a novel Ī²-sandwich fold with a similar shape to Ī²Ī³-crystallins and tryptophan RNA attenuation proteins. Because RIM undergoes fast and extensive degradation in vitro, its narrow cylindrical shape was rapidly measured by small-angle X-ray scattering before proteolysis could occur. The crystal structure of RIC comprises three tandem Ī²-sandwich domains similar to those in RII, but increasing in their hydrophobicity with proximity to the outer membrane. In addition, the key Ca\u3csup\u3e2+\u3c/sup\u3e ion that rigidifies the linkers between RII domains is not present between the first two of these RIC domains. This more flexible RI linker near the cell surface can act as a 'pivot' to help the 0.6-Ī¼m-long MpIBP sweep over larger volumes to find its binding partners. Since the physical features of RI are well conserved in the RTX adhesins of many Gram-negative bacteria, our detailed structural and bioinformatic analyses serve as a model for investigating the surface retention of biofilm-forming bacteria, including human pathogens.\u3c/p\u3
Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor
The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology
Membrane catalysis of peptideāreceptor bindingThis paper is one of a selection of papers published in this special issue entitled āCanadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting ā Protein Folding: Principles and Diseasesā and has undergone the Journal's usual peer review process.
Biophysical characterization of G-protein coupled receptorāpeptide ligand bindingThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting ā Membrane Proteins in Health and Disease, and has undergone the Journalās usual peer review process.
Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.
Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation
Periodicity in Structure, Bonding, and Reactivity for P-Block Complexes of a Geometry-Constraining Triamide Ligand
The use of pincer ligands to access non-VSEPR
geometries at main-group centers is an emerging strategy for eliciting new
stoichiometric and catalytic reactivity. As part of this effort, several
different tridentate trianionic substituents have to date been employed at a
range of different central elements, providing a patchwork dataset that
precludes rigorous structure-function correlation. Here we report an analysis
of periodic trends in structure (solid, solution, and gas phase), bonding, and
reactivity based on systematic variation of the central element (P, As, Sb, or
Bi) with retention of a single tridentate triamide substituent. In this
homologous series, the central element can adopt either a bent or planar
geometry. The tendency to adopt planar geometries increases descending the
group with the phosphorus triamide (1) and its arsenic congener (2)
exhibiting bent conformations, and the antimony (3) and bismuth (4)
analogues exhibiting a predominantly planar structure in solution. This trend
has been rationalized using the energy decomposition analysis. A rare
phase-dependent dynamic covalent dimerization was observed for 3 and the
associated thermodynamic parameters were established quantitatively. Planar
geometries were found to engender lower LUMO energies and smaller band gaps as
compared to bent ones, resulting in different reactivity patterns. These
results provide a benchmark dataset to guide further research in this rapidly
emerging area