William Royer - Academia.edu (original) (raw)

Papers by William Royer

resolved crystallography, Xe binding and computational analysis

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activat... more C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 −/− cells transfected with tetramer destabilizing mutants. Mutants that inhibit tetramer formation show a decrease in exp...

Journal of Biological Chemistry

Edited by Wolfgang Peti C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregu... more Edited by Wolfgang Peti C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD ؉-and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate-and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer. C-terminal binding proteins 1 and 2 (CtBP1 3 and CtBP2) are paralogous transcriptional co-regulators that modulate numerous cellular processes by binding transcription factors and recruiting chromatin remodeling enzymes such as histone deacetylases, methyl transferases, and demethylases to targeted promoters (1-3). CtBP1 was first identified through interactions with the C-terminal region of the adenovirus E1A oncoprotein and the ability to modulate E1A-transforming activities (4, 5). CtBP co-transcriptional function is important in normal embryogenesis, as it is a regulator of the epithelial-to-mesenchymal transition and is integral in proper fetal cell differentiation. Knockout experiments in mice reveal distinct roles for CtBP1 and CtBP2 in development, with the loss of CtBP2 embryonically lethal, whereas CtBP1-null mice are small but the majority survive (6). Alternate splice forms of CtBP1 and-2 also have nonnuclear roles, including membrane trafficking (7). Both CtBP paralogues have been implicated as global repressors of the epithelial phenotype and of apoptotic pathways (1), and numerous lines of evidence implicate human CtBP in cancer progression. CtBP is a corepressor of genes including tumor suppressive pro-apoptotic factors (Bik and Noxa), cytoskeletal/ cell adhesion molecules (keratin-8 and E-cadherin), and cellcycle inhibitors (2, 8). CtBP has also been found to act as a coactivator of growth and metastasis-related genes (Tiam1 and MDR1 and certain Wnt target genes), which facilitate the epithelial-to-mesenchymal transition (9-11). Consistent with its role in repression of apoptotic pathways and activation of growth and metastasis, CtBP is up-regulated in a number of cancer tissues, including colorectal cancer (12), melanoma (13), metastatic prostate cancer (14), esophageal squamous cell carcinoma (15), ovarian cancer (16), and breast cancer (17, 18). Strikingly, elevated levels of CtBP in tumor tissue have been correlated with poorer survival in breast cancer (19), ovarian cancer (16), and hepatocellular carcinoma (20). Recent results add to evidence of a link between CtBP and cancer progression by showing increased survival of APC min/ϩ mice when CtBP2 levels are lowered by CtBP2 ϩ/Ϫ heterozygosity (21). CtBP is unique among transcription factors in the incorporation of a D-isomer-specific 2-hydroxyacid dehydrogenase domain, which reduces or oxidizes substrates using the coenzyme NAD(P) ϩ /NAD(P)H (22, 23). The best substrate identified to date for CtBP is 4-methylthio-2-oxobutyric acid (also known as MTOB or 2-keto-4-methylthiobutyrate) (24), although whether this is a physiologically relevant substrate is unknown. Whereas evidence indicates that catalytic activity is not required for some CtBP activities (8, 25), mutant studies suggest that catalytic activity of CtBP can be important for Drosophila melanogaster development (26).

Biophysical Journal

Regulation of endothelial barrier function is critical for vascular homeostasis, as dynamic and l... more Regulation of endothelial barrier function is critical for vascular homeostasis, as dynamic and local control of vascular permeability permits macromolecular transport, immune surveillance, and deposition of a fibrin barrier to contain infection at sites of inflammation. Many of the signaling pathways promoting useful vascular permeability, however, are also triggered during disease, resulting in prolonged or uncontrolled vascular leak. Hyper-permeability triggered by inflammation or ischemia in the heart, brain, or lung promotes edema, exacerbates disease progression, and impairs recovery. Currently there is no clinical solution for the regulation of the endothelial barriers. A main reason for the lack of translational solutions is the technical difficulties (e.g. low throughput, and lengthy assays) to explore libraries of small compounds to begin drug development. Regulating endothelial barriers is crucial in the prevention of disease, disease progression, and the discovery and implementation of new therapies. Claudins, and occludin, are Tight Junction membrane proteins (TJ-MP) that seal the paracellular space creating the endothelial barriers. Our laboratory has devised a strategy to express TJ-MPs in the outer membrane of E. coli. TJ-MP expression drives cell-cell interactions above the unicellular behavior of E. coli. TJ-MPs force this aggregation in a manner that correlates with their strength in the native endothelial barriers. The aggregation of E. coli can be quantified using flow cytometry. Our preliminary results suggest that our assay has sensitivity to detect hyper-permeability (barrier opening) as well as hypo-permeability (barrier closing or strengthening).

Nature communications, Jan 28, 2017

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate v... more Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA ...

Bioorganic & medicinal chemistry, Jun 15, 2016

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expres... more C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues w...

Structure and Function of Invertebrate Oxygen Carriers, 1991

The crystal structures of the Hbs from the clam Scapharca inaequivalvis (Hbl, a homodimer, and Hb... more The crystal structures of the Hbs from the clam Scapharca inaequivalvis (Hbl, a homodimer, and Hbll, a heterotetramer) (1-3), and from the “fat innkeeper” worm Urechis caupo (a homotetramer) (4) have revealed novel assemblages of Mb-folded chains that differ markedly from that characteristic of the α2β2 vertebrate Hb tetramer (5).

The Journal of biological chemistry, Jan 25, 1988

X-ray diffraction data to a minimum Bragg spacing of 5.5 A have been collected from crystals of L... more X-ray diffraction data to a minimum Bragg spacing of 5.5 A have been collected from crystals of Lumbricus terrestris erthrocruorin, a 3.9 x 10(6)-dalton respiratory protein. Self-rotation function calculations from these data reveal D6 symmetry to a resolution of at least 6 A. These calculations show that erythrocruorin molecules pack in their crystals with molecular diads coincident with crystallographic diads along the a axis. Packing constraints limit the position of the molecular center to within 40 A of x = 1/4a.

Methods in Enzymology, 2008

Nature Structural & Molecular Biology, 2008

ACS chemical biology, Jan 30, 2015

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulato... more Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the c...

Structure, 2006

Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular ... more Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 Å crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D 6 symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein.

"Protein Engineering, Design and Selection", 1995

... Candace M^ummerford1A3, Animesh Pardanani1-2, Andrew H.Betts1*2, Anthony R.Poteete1-4, Gianni... more ... Candace M^ummerford1A3, Animesh Pardanani1-2, Andrew H.Betts1*2, Anthony R.Poteete1-4, Gianni Coiotti5 and William E.RoyerJr1-2&quot;6 ... Natl Acad. Sci. USA, 78, 4204-4208. Chiancone.E., Vecchini,P., Verzili.D., Ascoli.F. and Amonini,E. (1981) J. Mol. Biol., 152, 577-592. ...

Proceedings of the National Academy of Sciences, 1996

One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption o... more One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72 3 Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.

Journal of Molecular Biology, 1998

A cluster of interface ordered water molecules has been proposed to act as a key mediator of inte... more A cluster of interface ordered water molecules has been proposed to act as a key mediator of intersubunit communication in the homodimeric hemoglobin of Scapharca inaequivalvis. Mutations of Thr72 to Val and Ile, which lack the hydroxyl group to hydrogen bond the deoxy interface water molecules, result in sharply altered functional properties. We have determined the high resolution (1.6 ± 1.8 A Ê) crystal structures of these two mutants in both the deoxygenated and CO-liganded states. These structures show minimal protein structural changes relative to the same native derivatives, despite greater than 40-fold increases in oxygen af®nity. In the deoxy state of both mutants two water molecules at the periphery of the water cluster are lost, and the remaining cluster water molecules are destabilized. The CO-liganded structures show key differences between the two mutants including a more optimal interface packing involving Ile72 that acts to stabilize its high af®nity (R) state. This additional stabilization allows rationalization of its lowered cooperativity within the context of a two-state model. These studies support a key role of ordered water in cooperative functioning and illustrate how subtle structural alterations can result in signi®cantly altered functional properties in an allosteric molecule.

Journal of Molecular Biology, 1997

We have re®ned the crystal structure of deoxyhemoglobin S (b Glu6 3 Val) at 2.05 A Ê resolution t... more We have re®ned the crystal structure of deoxyhemoglobin S (b Glu6 3 Val) at 2.05 A Ê resolution to an R-factor of 16.5% (free R 21.5%) using crystals isomorphous to those originally grown by Wishner and Love. A predominant feature of this crystal form is a double strand of hemoglobin tetramers that has been shown by a variety of techniques to be the fundamental building block of the intracellular sickle cell ®ber. The double strand is stabilized by lateral contacts involving the mutant valine interacting with a pocket between the E and F helices on another tetramer. The new structure reveals some marked differences from the previously re®ned 3.0 A Ê resolution structure, including several residues in the lateral contact which have shifted by as much as 3.5 A Ê. The lateral contact includes, in addition to the hydrophobic interactions involving the mutant valine, hydrophilic interactions and bridging water molecules at the periphery of the contact. This structure provides further insights into hemoglobin polymerization and may be useful for the structurebased design of therapeutic agents to treat sickle cell disease.

Journal of Molecular Biology, 1987

Journal of Molecular Biology, 2004

Erythrocruorins are highly cooperative giant extracellular respiratory complexes found in annelid... more Erythrocruorins are highly cooperative giant extracellular respiratory complexes found in annelids, where they serve the same function as red blood cells. Our previous 5.5A resolution crystal structure of Lumbricus terrestris erythrocruorin revealed a hierarchical organization of 144 oxygen-binding hemoglobin chains that are assembled into 12 dodecamers arranged at the periphery of the complex around a central scaffold formed by 36 non-hemoglobin subunits. We present here the 2.6A resolution crystal structure of the Lumbricus hemoglobin dodecameric subassembly, which provides the first atomic models of the erythrocruorin allosteric core. The hemoglobin dodecamer has a molecular 3-fold axis of symmetry that relates three heterotetramers, each of which is composed of two tightly associated heterodimers. The structure reveals details of the interfaces, including key side-chain interactions likely to contribute to ligand-linked allosteric transitions, and shows the crowded nature of the ligand-binding pockets. Comparison of the Lumbricus dimeric assemblies with similar ones from mollusks and echinoderms suggests plausible pH-dependent quaternary transitions that may occur in response to proton binding and ligand release. Thus, these results provide the first step towards elucidating the structural basis for the strong allosteric properties of Lumbricus erythrocruorin.