Structural Biology Core Facility Publications

Read about the science being performed at Brown University's Structural Biology Core Facility.

Burke KA, Janke AM, Rhine CL, and Fawzi NL. "Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II" Mol Cell. 2015, (online ahead of print).

The N-terminal domain of the RNA-binding protein Fused in Sarcoma (FUS) plays a role in amyotrophic lateral sclerosis and frontotemporal dementia. Brown University postdoctoral fellow Kathleen Burke, graduate student Christy Rine, and undergraduate Abigail Jahnke from the lab of Dr. Nicolas Fawzi investigated the behavior of this domain using the SBCF 850 MHz NMR and demonstrated that it was disordered in solution. They also observed that as the protein concentration increased, FUS formed phase-separated droplets. Both RNA and RNA polymerase II facilitated the formation of these droplets. Unlike solid inclusions such as amyloid plaques, the droplets are dynamic, and FUS diffuses freely within them. Thanks to the high sensitivity of the 850, Burke and colleagues were able to demonstrate that FUS is still disordered and flexible even inside the droplets. This dynamic character is unique and may have important physiological implications.

Krishnan N, Krishnan K, Connors CR, Choy MS, Page R, Peti W, Van Aelst L, Shea SD, and Tonks NK. "PTP1B inhibition suggests a therapeutic strategy for Rett syndrome" J Clin Invest. 2015, 125(8):3163-77.

Choy MS, Yusoff P, Lee IC, Newton JC, Goh CW, Page R, Shenolikar S, and Peti W. "Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase" Cell Rep. 2015, 11(12):1885-91.

Conicella A, and Fawzi NL. "The C-Terminal Threonine of Aβ43 Nucleates Toxic Aggregation via Structural and Dynamical Changes in Monomers and Protofibrils" Biochemistry 2014, 53 (19): 3095-3195.

The aggregation of amyloid-β (Aβ) peptides into plaques or soluble oligomers is believed to cause Alzheimer's disease. However, many different Aβ peptides are present in the brain. The most common are Aβ40 and Aβ42, but some studies have suggested that other forms of the peptide may be the trigger for aggregation and disease. Aβ43 is one of these suspected culprits, because it is significantly enriched in plaques and is more toxic than Aβ42. Using the 850 MHz NMR spectrometer of the Structural Biology Core Facility, Brown University graduate student Alex Conicella and Professor Nick Fawzi studied the dynamics of Aβ43 in order to understand the cause of this enhanced toxicity. They found that the Aβ43 peptide formed protofibrils faster than Aβ42, and at lower concentrations. They also determined that Aβ43 is more rigid at its C-terminus than either of the other two peptides, as well as being very similar to the aggregation-prone Aβ42 when in protofibrils. These data support the hypothesis that Aβ43 serves as a trigger for the formation of toxic assemblies of Aβ and thus plays a significant role in the pathology of Alzheimer's.

Krishnan N, Koveal D, Miller DH, Xue B, Akshinthala SD, Kragelj J, Jensen MR, Gauss C-M, Page R, Blackledge M, Muthuswamy SK, Peti W and Tonks N. "Targeting the disordered C terminus of PTP1B with an allosteric inhibitor." Nature Chem. Biol. 2014 (ahead of print)

Protein-tyrosine phosphatase 1B (ptp1b) is a signaling protein that plays a role in a variety of pathways associated with diseases including cancer, diabetes, and obesity. As a result there has been a great deal of interest in developing inhibitors of this enzyme. However, the nature of the chemistry it performs has proven a significant barrier to creating effective drugs. Brown University students Dorothy Koveal and Dan Miller, with Professors Wolfgang Peti and Rebecca Page, collaborated with research teams at the University of Toronto and Institut de Biologie Structurale in Grenoble to characterize an inhibitor, MSI-1436, that interferes with ptp1b function without binding to the active site. Like most enzymes, ptp1b performs catalysis with a folded domain, but it also has a large C-terminal region that serves a regulatory function and is predominantly unfolded. Using the NMR spectrometers of the Structural Biology Core Facility, the Page and Peti labs were able to show that this C-terminal region, though disordered, possesses residual secondary structure. They were also able to identify two specific sites where MSI-1436 binds. One of these sites lay within one of the predicted helices, and mutations to disrupt the helical structure significantly reduced the inhibitor's affinity. The second binding location also lies within the unfolded domain, but near another allosteric regulation site.  Although the mechanism of inhibition is not yet clear, this result suggests promising new avenues for controlling ptp1b activity.

Choy MS, Hieke M, Kumar GS, Lewis GR, Gonzalez-Dewhitt KR, Kessler RP, Stein BJ, Hessenberger M, Nairn AC, Peti W, and Page R. "Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code." Proceedings of the National Academy of Sciences 2014, 111 (11): 4097-4102.

The Brown University labs of Rebecca Page and Wolfgang Peti collaborated on an investigation of serine-threonine protein phosphatase 1 (PP1). PP1 is a catlytic unit involved in hundreds of different dephosphorylation reactions involved in numerous regulatory pathways, including processes as diverse as carbohydrate metabolism and cell-cycle progression. It gains specificity for each pathway by binding to other proteins to form a phosphatase holoenzyme, although it has been difficult to predict how any given protein will bind PP1 due to the vast diversity of regulators. One such partner is PNUTS, which plays a key role in many processes of the cell nucleus. Of special interest, the PP1:PNUTS holoenzyme regulates two tumor suppressors: p53 and Rb. Using NMR, the research team identified the smallest piece of PNUTS that encompassed the whole PP1 binding site. Then they used crystallography to determine the structure of this PP1:PNUTS complex. The new structure demonstrated that PNUTS binds to the same part of PP1 that Rb does, but also to two other sites, generating a high-affinity interaction that only breaks down once PNUTS gets phosphorylated or knocked down in concentration. These results not only illuminate the competition between PNUTS and Rb for PP1 binding, but also revealed additional structural motifs related to PP1 binding. This will allow researchers to predict the binding behavior of up to a quarter of known PP1 regulators.

Kumar GS, Zettl H, Page R, and Peti W. "Structural Basis for the Regulation of the MAP Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in SolutionJournal of Biological Chemistry 2013, 288: 28347-56

Brown University postdoctoral associates Senthil Kumar and Heiko Zettl, from the Page-Peti lab, used NMR spectroscopy to figure out how DUSP16 interacts with p38α. DUal-Specificity Phosphatases (DUSPs) regulate MAP kinases, and so are sometimes called MKPs (MAP Kinase Phosphatases). Like the KIM-PTPs, they primarily bind via a short amino acid sequence known as the KIM, but in DUSPs the KIM is part of a completely folded MAP Kinase Binding Domain (MKBD). Dr. Kumar showed that DUSP16 binds more strongly to p38α than DUSP10, another member of the protein family. Using NMR experiments performed on the Structural Biology Core Facility 500 and 850 MHz spectrometers, he found that this heightened affinity was related to an expanded binding site that inolved not only the KIM and nearby helices (as in DUSP10), but also interactions at an additional helix. These results indicate that there are key differences in binding between different DUSPs, explaining their different physiological roles and suggesting that it is likely possible to specifically disrupt particular MAP-DUSP complexes.

Francis DM, Kumar GS, Koveal D, Tortajada A, Page R and Peti W. "The Differential Regulation of p38α by the Neuronal Kinase Interaction Motif Protein Tyrosine Phosphatases, a Detailed Molecular Study." Structure 2013, 21 (9): 1612-1623

Brown University graduate students Dana Francis and Dorothy Koveal, and postdoctoral associate Senthil Kumar, from the Page-Peti lab, used NMR spectroscopy, Small-Angle X-ray Scattering (SAXS), Isothermal Titration Calorimetry (ITC), and HADDOCK modeling to identify the structural differences in the interaction of p38α with the family of KIM protein tyrosine phosphatases (KIM-PTPs), which have a catalytic domain and a disordered region that contains a Kinase Interaction Motif. KIM-PTPs regulate the activity of MAP kinases like p38α, which is implicated in inflammatory responses and autoimmune disease. This study focused on PTPSL and STEP. Using NMR spectroscopy and SAXS, the Page-Peti team found that PTPSL binds to p38α primarily using the KIM and another unfolded sequence known as the KIS, and that the complex formed in solution is extended, similarly to the binding interaction they described previously for a KIM-PTP known as HePTP. They found that the interaction of STEP, a drug target in Alzheimer's disease, was very different. SAXS data clearly show that the STEP-p38α complex is compact, and the NMR data indicated that unlike the other KIM-PTPs studied, STEP's catalytic domain interacts directly with p38α.  This difference in binding modes explains the lower catalytic efficiency of STEP, and offers novel approaches for controlling the activity of p38α.

Koveal D, Clarkson MW, Wood TK, Page R, and Peti W. "Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas  aeruginosa" Journal of Molecular Biology 2013, 425 (12); 2219-2231.

Brown University graduate student Dorothy Koveal, from the Page-Peti lab, used NMR spectroscopy to determine the structure of a phosphatase that controls biofilm formation in P. aeruginosa in isolation and in complex with phosphate. Using the 500 MHz NMR spectrometer at Brown University, she also obtained information about the dynamics of the protein's backbone amide groups. These experiments showed that the unbound protein had significantly greater flexibility in its active site than the phosphate-bound protein. This restraint of motion may be related to enzymatic activity or substrate specificity.

Koveal D, Schuh-Nuhfer N, Ritt D, Page R, Morrison DK, and Peti W. "A CC-SAM, for Coiled-Coil Sterile-α Motif, Domain Targets the Scaffold KSR-1 to Specific Sites in the Plasma Membrane" Science Signaling 2012, 5 (255): ra94.

Brown University graduate student Dorothy Koveal, from the Page-Peti lab, used NMR spectroscopy to determine the structure of a novel domain fusing a coiled-coil motif to a sterile-α motif. With the assistance of collaborators at the National Cancer Institute, she found that this domain targeted the scaffolding protein KSR-1 to membrane ruffles in vivo. Using the Brown University 500, she determined that CC-SAM underwent a structural rearrangement in the presence of specific kinds of lipid micelles and bicelles that explained the domain's targeting capabilities.