Crystal structure of the p53 tumor suppressor bound as a tetramer to DNA. The tumor suppressor p53 regulates downstream genes in response to many cellular stresses and is frequently mutated in human cancers.  We used a crosslinking strategy to trap a tetrameric p53 core domain bound to DNA and reported its X-ray crystal structure [3EXJ, 3EXL].  The tetramer can bind to straight B-form DNA and monomer-dimer contacts play more significant roles than dimer-dimer contacts in p53 binding to DNA.  Analysis of the dimer-dimer contacts and conserved residues leads to the hypothesis that multiple tetramerization modes are possible.

Oncogene 2009, 28, 325-33.

Structure of the protein acetyltransferase from Sulfolobus Solfataricus. The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA binding protein, on K16 to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a “primitive” model for chromatin regulation analogous to histone modification in eukaryotes. We determined the crystal structure of PAT in complex with coenzyme A [3F8K]. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional “bent helix” proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate limiting. The catalytic efficiency of PAT towards ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

J. Biol. Chem. 2009, 284, 19412-9.

Structure of the FOXO1 transcription factor bound to DNA. FoxO transcription factors regulate the transcription of genes that control metabolism, cellular proliferation, stress tolerance and possibly lifespan. A number of post-translational modifications within the forkhead DNA binding domain regulate FoxO mediated transcription. We determined the crystal structures of FoxO1 bound to three different DNA elements [3C06, 3C07, 3COA] and measured the change in FoxO1-DNA affinity with acetylation and phosphorylation. The structures revealed additional contacts and increased DNA distortion for the highest affinity DNA site. The flexible wing 2 region of the forkhead domain was not observed in the structures but is necessary for DNA-binding, and we showed that p300 acetylation in wing 2 reduces DNA affinity. We also showed that MST1 phosphorylation of FoxO1 prevents high affinity DNA binding. The observation that FoxO-DNA affinity varies between response elements and with post-translational modifications suggests that modulation of FoxO-DNA affinity is an important component of FoxO regulation in health and misregulation in disease.

Structure 2008, 16, 1407-1416.

Development of novel p21-activated kinase inhibitors. p21-activated kinases (or PAKs) have elevated activity in several different cancers and have therefore become attractive drug targets. Employing novel organometallic kinase inhibitor libraries, we have identified lead compounds that show strong selectivity for the PAKs in the low-micromolar to mid-nanomolar range. We have also obtained crystal structures of these inhibitors bound to PAK1 [3FYO, 3FXZ] and are using these structures for structure-based inhibitor optimization towards isoform selective PAK inhibitors.

J. Am. Chem. Soc. 2008, 130, 15764-5.

Structure of the Rtt109 histone acetyltransferase. Rtt109/KAT11 is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genomic stability.  Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity.  We determined the crystal structure of an Rtt109-AcCoA complex [3D35] and carried out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109–Vps75 complex and studies of Rtt109 function in vivo.  The Rtt109 structure reveals striking homology to the metazoan p300/CBP HAT domain, but exhibits functional divergence including atypical catalytic properties and mode of cofactor regulation.  The structure also reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells.

Nat. Struc. Mol. Biol. 2008, 3, 305-316.

The Gal4/DNA complex revisited. Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that binds DNA as a homodimer and can activate transcription by interacting with the mutant Gal11P protein. Although structures have been reported of the Gal4 dimerization domain and the binuclear cluster domain monomer bound to DNA as a dimer, the structure of the “complete” Gal4 dimer bound to DNA has not previously been described. We determined the structure of a complete Gal4 dimer bound to DNA [3COQ]additional biochemical studies to address the molecular basis for Gal4 dimerization in DNA binding. We find that Gal4 dimerization on DNA is mediated by an intertwined helical bundle that deviates significantly from the solution NMR structure of the free dimerization. Associated biochemical studies show that the dimerization domain of Gal4 is important for DNA binding and protein thermostability.  We also map the interaction surface of the Gal4 dimerization domain with Gal11P.

Structure 2008, 16, 1019-1026

The retinoblastoma protein bound to adenovirus E1A. The Adenovirus (Ad) E1A oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the pRb tumor suppressor. We determined the crystal structure of the pRb pocket domain in complex with Conserved Region 1 (CR1) of Ad5-E1A [2R7G].  The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin-folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain employ similar conserved non polar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.  

Genes Develop. 2007, 21, 2711-2716.

Human monocytic leukemia zinc-finger histone acetyltransferase. The human monocytic leukemia zinc-finger (MOZ) protein is an essential transcriptional coactivator and histone acetyltransferase (HAT) that plays a primary role in the differentiation of erythroid and myeloid cells and that is required to maintain hematopoietic stem cells.  Chromosomal translocations involving the HAT-encoded region are also associated with acute myeloid leukemia (AML).  We determined the X-ray crystal structure of the MOZ HAT domain [2RC4] and carried out related biochemical studies.  We find that the HAT domain contains a central region that is structurally and functionally conserved with the yeast MYST HAT protein Esa1, but contains more divergent N- and C-terminal regions harboring a TFIIIA-type zinc finger (purple) and helix-turn-helix (red) DNA-binding motifs  Solution DNA-binding and acetyltansferase activity assays, in concert with mutagenesis, confirm that the MOZ HAT domain binds strongly to DNA through the zinc finger and helix-turn helix motifs and that DNA binding and catalysis is not mutually exclusive. Consistent with the DNA-binding properties of MOZ, we also show that MOZ is able to acetylate nucleosomes and free histones equally well; while other HATs prefer free histones.  Our results reveal, for the first time, that enzymatic and DNA targeting activities can be contained within the same chromatin regulatory domain.

J. Biol. Chem. 2007, 282, 36603-36613.

The p300 transcriptional coactivator. The transcriptional coactivator p300/CBP is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors, and dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. We determined a high resolution X-ray crystal structure of a semisynthetic heterodimeric p300 HAT domain in complex with a bisubstrate inhibitor, Lys-CoA [3BIY].  This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs but reveals a number of novel features that explain the broad substrate specificity and preference for nearby basic residues.  Based on this structure and accompanying biochemical data, we propose that p300/CBP employs an unusual “hit-and-run” (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs.  A number of disease-associated mutations can also be readily accounted for by the p300 HAT structure.  These studies pave the way for novel epigenetic therapies involving modulation of p300/CBP HAT activity.

Nature, 2008, 451, 846-850.

Ubiquitin recognition by the ovarian tumor domain protein. Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization.  Deubiquitinating enzymes (DUBs) are proteases that reverse this ubiquitination.  Previous studies demonstrate that proteins that contain an Ovarian Tumor (OTU) domain possess deubiquitinating activity.  This domain of about ~130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals.  We carried out functional studies on the OTU domain containing protein from yeast, Otu1.  We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with K48 linkages, having little or no activity on K63- and K29-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-Associated Degradation (ERAD) pathway. This interaction involves an N-terminal ubiquitin fold domain and results in reduced DUB activity of Otu1.  We also determined the X-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin [3BY3, 3C0R] and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases.  Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain containing proteins.

J. Biol. Chem., 2008, 283, 11038-11049.

Structure-based design of an organoruthenium Phosphatidyl-Inositol-3-Kinase inhibitor. Mutations that constitutively activate the PI3K signaling pathway, including alterations in PI3K, PTEN and AKT are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. We report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3Kg/E5E2 complex [3CSF, 3CST] reveals the molecular features that give rise to this potency and selectivity towards lipid kinases with implications for the design of a subsequent generation of PI3K-isoform specific organometallic inhibitors.

ACS Chem. Biol., 2008 , 3, 305-316.

Structure of the Bre5 cofactor bound to the Ubp3 deubiquitinating enzyme. Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell.  A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit.  We have determined the X-ray crystal structure of an Ubp3/Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer (green/yellow) interacts with two L-shaped b-strand-turn-a-helix motifs of Ubp3 (blue/red) [1QIY].  The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through anti pllel b-arasheet hydrogen bonds and van der Waals contacts.  Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo.  Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3/Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.

J. Mol. Biol. 2007, 372, 194-204.

Structure of a sirtuin protein bound to nicotinamide. The Sir2 family of proteins are broadly conserved NAD+-dependant deacetylases that are implicated in diverse biological processes including DNA regulation, metabolism and longevity.  Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base exchange reaction to reform NAD+ at the expense of deacetylation.  To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide (yellow) bound to a Sir2 homologue, yeast Hst2, in complex with its acetyl-lysine 16 histone H4  substrate (green) and a reaction intermediate analog, ADP-HPD (purple) [2OD2, 2OD7, 2OD9 ].  Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base exchange site that is distinct from the so-called “C pocket” binding site for the nicotinamide group of NAD+.  These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

Mol. Cell 2007; 25: 463-472.

Structure of the p53 core domain bound to a small molecule stabilizing compound. The p53 transcriptional regulator is the most frequently mutated protein in human cancer and the majority of tumor-derived p53 mutations map to the central DNA-binding core domain, with a subset of these mutations resulting in reduced p53 stability.  We have determined the 1.55 Å crystal structure of the mouse p53 core domain with a molecule of tris(hydroxymethyl)aminomethane (Tris) [2IOI, 2IOO, 2IOM ] bound through multiple hydrogen bonds to a region of p53 shown to be important for repair of a subset of tumor-derived p53 stability mutations.  Consistent with the hypothesis that Tris binding stabilizes the p53 core domain, we present equilibrium denaturation experiments that demonstrate that Tris binding increases the thermodynamic stability of the mouse p53 core domain by 0.74 kcal/mol; and molecular dynamic simulations revealing an overall reduction in root-mean-square deviations of the core domain of 0.7 Å when Tris is bound.  We also show that these crystals of the p53 core domain are suitable for Multiple Solvent Crystal Structure approach to identify other potential binding sites for possible core domain stabilization compounds.  Together, these studies provide a molecular scaffold for the structure-based design of p53 stabilization compounds for development as possible therapeutic agents.

Acta Cryst.2006; 62: 1484-1493.

Structure of a human ASF1a/HIRA histone chaperone complex. Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We determined the crystal structure of an ASF1a/HIRA heterodimer [2I32]and carried out a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B-domain forms an antiparallel b-hairpin that binds perpendicular to the strands of the b-sandwich of ASF1a, via b-sheet, salt-bridge and van der Waals contacts. The N- and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B-domain. CAF-1 p60 also employs B-domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes.

Nature Struc. Mol. Biol. 2006; 13: 921-929.

Structure of the p53 core domain dimer bound to DNA. The p53 tumor suppressor protein binds to DNA as a dimer of dimers to regulate transcription of genes that mediate responses to cellular stress.  We have prepared a crosslinked-trapped p53 core domain dimer bound to decamer DNA and have determined its structure by X-ray crystallography to 2.3 Å resolution [2GEQ].  The p53 core domain subunits bind nearly symmetrically to opposite faces of the DNA in a head-to-head fashion with a loop-helix motif making sequence-specific DNA contacts and bending the DNA by about 20° at the site of protein dimerization.  Protein subunit interactions occur over the central DNA minor groove and involve residues from a zinc-binding region.  Analysis of tumor derived p53 mutations reveals that the dimerization interface represents a third hot-spot for mutation that also includes residues associated with DNA contact and protein stability.  Residues associated with p53 dimer formation on DNA are poorly conserved in the p63 and p73 paralogs, possibly contributing to their functional differences. 

J. Biol. Chem. 2006; 281: 20494-20502.

Structure of a Leu3/DNA complex. Gal4 is the prototypical Zn2Cys6 binuclear cluster transcriptional regulator that binds as a homodimer to DNA containing inverted CGG half-sites.  Leu3, a member of this protein family binds to everted (opposite polarity to inverted) CGG half-sites and an H50C mutation within the Leu3 Zn2Cys6 binuclear motif abolishes its transcriptional repression function without impairing DNA binding.  We determined the X-ray crystal structures of DNA complexes with Leu3 and Leu3(H50C) and [2ERG, 2ERE, 2ER8 ] solution DNA-binding studies of selected Leu3 mutant proteins.  These studies reveal the first molecular details of everted CGG half-site recognition and suggest a role for the H50C mutation in transcriptional repression.  Comparison with the Gal4/DNA complex shows an unexpected conservation in the DNA recognition mode of inverted and everted CGG half-sites and points to a critical function of a linker region between the Zn2Cys6 binuclear cluster and dimerization regions in DNA-binding specificity.  The broader implications of these findings are discussed.

Structure 2006; 14: 725-735.

Structure of the Snf1/AMPK kinase domain. The Snf1/AMPK kinases are intracellular energy sensors and the AMPK pathway has been implicated in a variety of metabolic human disorders.  We determined the crystal structure of the kinase domain from yeast Snf1 [2FH9], revealing a bi-lobe kinase fold with greatest homology to cyclin-dependant kinase-2.  Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution as demonstrated by equilibrium sedimentation and in yeast cells as shown by coimmunoprecipitation of differentially tagged intact Snf1.  A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases.  The conformation of the conserved aC helix, and the burial of the activation segment and substrate-binding site within the dimer suggests that it represents an inactive form of the kinase.  Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family and an avenue for the development of AMPK-specific activating compounds.

Structure 2006; 14: 477-485.

Structure and function of the SWIRM domain. The SWIRM domain is a novel module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. We determined the high-resolution crystal structure of the SWIRM domain from Swi3 [2FQ3] and characterize the in vitro and in vivo function of the SWIRM domains from Swi3 and Rsc8. The Swi3 SWIRM forms a 4-helix bundle containing a pseudo 2-fold axis, and a helix-turn-helix motif commonly found in DNA binding proteins. We also show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multi-functional module for the regulation of gene expression.

Proc. Natl. Acad. Sci. USA 2006, USA 2006, 103, 2057-2062.

Structure of the Human Papillomavirus E7 Oncoprotein. The E7 oncoprotein from human papillomavirus (HPV) mediates cell transformation, in part, by binding to the human pRb tumor suppressor protein and E2F transcription factors, resulting in the dissociation of pRb from E2F transcription factors and the premature cell progression into the S-phase of the cell cycle. This activity is mediated by the LXCXE motif and the CR3 zinc-binding domain of the E7 protein. We determined the X-ray crystal structure of the CR3 region of HPV E7 [2B9D] and employed structure-based mutagenesis to investigate its mode of pRb/E2F complex disruption. The structure reveals a novel zinc bound E7-CR3 obligate homodimer that contains 2 surface patches of sequence conservation and mutational analysis reveal that these patches are required for pRb and E2F binding and pRb/E2F complex disruption. These studies provide an avenue for developing small molecule compounds that inhibit HPV-E7-mediated cell transformation.

J. Biol. Chem. 2005; 281: 578-586.

Structure of the NTF2 domain of the Bre5 deubiquitination cofactor. The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3 and it contains an NTF2-like protein recognition module that is essential for Ubp3 activity. We determined the X-ray crystal structure of the Bre5 NTF2-like domain [1ZX2] that reveals a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveals that under non-reducing conditions the Bre5 NTF2-like domain is exclusively dimeric while a disulfide bond deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the mid-nanomolar range, suggesting that dimer formation, and possibly also disulfide bond formation, may modulate Bre5 function in vivo. The structure was used as a scaffold to map the binding site for Ubp3. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding.

J. Biol. Chem. 2005; 280: 29176-29185 .

Structure of the yeast Hst2 homolog of the Sir2 histone deacetylase in ternary complex with histone peptide and NAD analogues. Yeast Hst2 (yHst2) is a member of the Sir2 family of NAD⁺-dependant protein deacetylases that are implicated to play roles in transcriptional silencing, DNA repair, genome stability, longevity, type-II diabetes and longevity. To define the mechanism of Sir2 activity, we determined the ternary structure of a Sir2 protein in complex with a histone H4 peptide and several NAD⁺ analogues [1Q1A, 1SZC, 1SZD] as well as binary [1Q17] and nascent [1Q14] structures. A comparison between the structures reveals a detailed mechanism for NAD⁺ and acetyl-lysine binding and hydrolysis and suggests avenues for the design of Sir2 regulatory molecules.

Structure 2003; 11: 1403-1411.
Proc. Natl. Acad. Sci. USA; 2004 101: 8563-8568
J. Mol. Biol. 2004; 337; 731-741

Nature Structural Biology 2003; 10: 864-871.

Molecular Cell 2003; 12: 461-473.

Biochemistry 2003; In press.

Structure of Tetrahymena GCN5 bound to a bisubstrate inhibitor. [1M1D] Histone acetyltransferases (HATs) have been implicated in cancer and therefore HAT-specific inhibitors may have therapeutic applications. The structure of the GCN5 HAT bound to a H3 peptide-coenzyme A conjugate shown here provides the first structural scaffold for the design of HAT-specific inhibitors. Surprisingly, the structure also reveals that the H3-peptide portion of the inhibitor is bound outside of the binding site for the histone substrate and that only 5 of the 20 amino acid residues of the inhibitor are ordered. Mutational and enzymatic data support the hypothesis that the observed structure corresponds to a late catalytic intermediate.

Proc. Natl. Acad. Sci. USA 2002; 99: 14065-14070.

Structure of the yeast Esa1 histone actyltransferase. Yeast Esa1 is a member of the MYST family of histone acetyltransferases (HATs) that use acetyl-CoA to acetylate specific lysine residues within histones to regulate gene expression. The structure of a Esa1-CoA complex, reveals structural similarity to the catalytic core of the GCN5/PCAF HAT family [1FY7]. More recent structural ([1MJ9], [1MJA], [1MJB]) and functional studies on Esa1 demonstrates that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This cysteine residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT family. Surprisingly, this mode of catalysis differs dramatically from the GCN5/PCAF HAT family, which mediates direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated histone lysine substrate. These results demonstrate that different HAT families can employ distinct catalytic mechanisms, and have implications for their distinct biological roles and for the development of HAT-specific inhibitors.

Mol. Cell 2000; 6: 1195-1207.
Nat. Struct. Biol. 2002: 862-869.

Structure of human p18INK4c. [1IHB] p18INK4c is a member of the INK4 family of proteins that regulate the G1 to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin dependant kinases 4 and 6. The p16INK4a member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16INK4a mutations reduce the protein's thermodynamic stability. Based on this observation, we used p18INK4c as a model to prepare INK4 protein mutants (highlighted in the attached figure) with increased thermal stability and enhanced cell cycle inhibitory activity ([1MX2], [1MX4], [1MX6]). These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16INK4a-mediated cancers.

Nat. Struct. Biol. 1998; 5: 74-81
J. Biol. Chem. 2002; 277: 48827-48833

Structure of the ternary SAP-1/SRF/c-fos SRE DNA complex. [1K6O]
Combinatorial DNA binding is a common mode of DNA regulation in eukaryotic organisms. An example of such combinatorial regulation occurs at the promoter of the c-fos proto-oncogene where the Ets protein, SAP-1, and the serum response factor (SRF) form a ternary complex with DNA to promote gene activation. The structure of the ternary complex reveals how SAP-1 (blue) and the SRF dimer (green) cooperate to recognize their respective DNA sequences (green) with the c-fos DNA (red).

J. Mol. Biol. 2001; 314: 505-516.

Structure of p53 core domain. [1HU8] The p53 tumor suppressor is a sequence-specific DNA binding protein that activates transcription in response to DNA damage to promote cell cycle arrest or apoptosis. The p53 protein is mutated in a majority oh human cancers and nearly all of the tumor-derived mutations map to the central core DNA-binding domain. The 2.7Å crystal structure of the mouse p53 core domain shows a non-crystallographic trimer with three nearly identical subunit-subunit (dimer) contacts. These studies have implications for how p53 undergoes structural rearrangement for sequence-specific DNA binding.

J. Biol. Chem. 2001; 276: 12120-12127.

Structure of the Elk-1/E74DNA complex. [1DUX] Elk-1 is a member of a large group of eukaryotic transcription factors that contain a conserved ETS DNA-binding domain and that cooperate with the serum response factor (SRF) to activate transcription of the c-fos protooncogene. Shown is the crystal structure of the Elk-1/E74DNA complex viewed from the C-terminal end of the a3 DNA recognition helix. The a-helices are colored in light blue and b-strands are colored in yellow. The DNA is colored in red and GGA core recognition sequence in green. The two residues distal from the DNA-binding surface, Asp 69 and Asp 38, which are found to affect the DNA-binding properties, are colored in light green.

Nat. Struct. Biol. 2000 Apr;7(4):292-7.

Structure of the BTB/POZ domain of PLZF. [1CS3] The evolutionarily conserved BTB/POZ domain from the PLZF (promyelocytic leukemia zinc finger) oncoprotein mediates transcriptional repression through the recruitment of corepressor proteins containing histone deacetylases in APL (acute promyelocytic leukemia). Shown is the crystal structure of the protein dimer with one subunit shown in green and the other subunit shown in blue.

Cancer Res. 1999; 59: 5275-82.

Structure of the HAT domain from human P/CAF transcriptional coactivator bound to coenzyme-A (PCAF/CoA). [1CM0] The crystal structure is shown as a ribbon diagram (green) with a transparent surface representation of the protein (white) with the bound CoA cofactor (yellow). Single and triple alanine mutations in the homologous yeast GCN5 protein that disrupt HAT activity are mapped in purple onto the protein surface and highlight the histone substrate binding site.

EMBO J. 1999; 18 : 3521-32.

Structure of the nascent HAT domain from the yeast GCN5 (yGCN5) transcriptional coactivator protein. [1YGH] A schematic of the structure is shown. The four domains of the protein are color-coded; the structurally conserved domain that makes up the structurally conserved core of the GCN5-related N-acetyltransferases (GNATs) is colored blue (motifs A and D), motif B that shows sequence but not structural homology within the GNATs is colored aqua, and the structurally variable N-terminal and C-terminal flanking regions are colored red and green, respectively.

Proc. Natl. Acad. Sci. USA 1999; 96: 8931-6

Structure of the DNA-binding domain of the yeast HAP1 transcriptional activator bound to a target DNA sequence from the UAS of the CYC7 promoter. [1HWT] The HAP1 protein is a member of the GAL4 family of fungal transcription factors that contain a conserved Zn2Cys6 binuclear cluster domain and that bind as homodimers to DNA targets containing 2 conserved DNA half-sites. The crystal structure reveals how the HAP1 homodimer adopts a dramatically asymmetric structure to bind the UASCYC7 sequence that contains direct repeats of CGG half sites (green). The Zn atoms that stabilize the Zn2Cys6 domains are shown in yellow. The crystal structures of DNA-complexes with HAP1 mutant proteins, (HAP1-18) [2HAP] and HAP1-PC7 [1QP9], that have aberrant transcriptional activities have also been determined.

Nat. Struc. Biol. 1999; 6: 64-7.
Nat. Struct. Biol. 1999; 6: 22-7.
Nucleic Acids Res. 2000 Oct 15;28(20):3853-63.

Structure of the ETS-domain of human SAP-1 bound to a high affinity target DNA sequence from the drosophilae E74 promoter [1BC8] and bound to an natural target DNA sequence from the human c-fos promoter. [1BC7] Protein-DNA interactions that are mediated by the DNA-recognition helix of SAP-1 are shown. Hydrogen bounds and van der Waals interactions are shown in as green and blue dotted lines, respectively. Water molecules that mediate protein-DNA contacts are shown in purple and the GGA core DNA sequence that is recognized by all ETS proteins is highlighted in green. The right view shows the E74 complex and the left view shows the c-fos complex.

Mol. Cel. 1998; 2: 201-12.