Michael A. Weiss

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation

SH2 (src‐homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein‐effector coupling is provided by the association of phosphatidylinositol 3‐kinase (PI 3‐kinase) with specific phosphorylation sites within the PDGF receptor, the c‐Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS‐1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3‐kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor‐, IRS‐1‐ and c‐Src‐derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3‐kinase. Although phosphotyrosine and both activating and non‐activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non‐local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS.

TRANSCRIPTIONAL elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex1—4. In prokaryotes its regulation pro-vides an important mechanism of genetic control1. Analogous eukaryotic mechanisms are not well understood5, but may control expression of proto-oncogenes6,7 and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved euk-aryotic transcriptional elongation factor TFIIS9 enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins10—14. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through10 and possibly nascent transcript cleavage11—15. Here we describe the three-dimensional NMR16 structure of a Cys4 nucleic-acid-binding domain from human TFIIS9—10. Unlike previously characterized zinc modules17—21, which contain an a-helix, this structure consists of a three-stranded β-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA trans-actions22—24, including RNAPII itself25. This new structure, desig-nated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures26 and highlights the growing recognition of the β-sheet as a motif of nucleic-acid recognition27—28.

RNA recognition by arginine-rich peptide motifs.

A ubiquitious class of RNA‐binding proteins is distinguished by an arginine‐rich motif. Such proteins function in transcription, translation, RNA trafficking, and packaging. Peptide models are derived from viral regulatory proteins, including the virulence factors Tat and Rev of mammalian immunodeficiency viruses. Structures of model peptide–RNA complexes exhibit diverse strategies of recognition based in each case on structural transitions. Induced RNA structures contain noncanonical elements such as purine–purine mismatches, base triples, and flipped bases. Such elements enlarge and extend the RNA major groove to create specific peptide‐binding pockets and surfaces. The repertoire of bound peptide structures—β‐hairpin, α‐helix, and helix–bend–helix—reflects the diversity of induced RNA architectures. This repertoire, reminiscent of primordial exon‐encoded peptides, may recapitulate early events in the transition between RNA and protein worlds. Peptide‐directed changes in modern RNA structures can provide a mechanism of signaling in higher‐order RNA–protein assemblies.

Evidence for a Physical Association between the Shc-PTB Domain and the βc Chain of the Granulocyte-Macrophage Colony-stimulating Factor Receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific α chain and a β common chain (βc) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. βc plays an essential role in the transduction of extracellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including βc) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the βc cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-βc chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of βc and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of βc (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between βc and Shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with βc. These observations provide an explanation for the previously described requirement for Tyr577 of βc for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.

High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases

BACKGROUND Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

Effects of patient size on radiation dose reduction and image quality in low-kVp CT pulmonary angiography performed with reduced IV contrast dose

The purpose of the study is to evaluate image quality and radiation exposure as a function of patient size for CT pulmonary angiography (CTPA) performed at reduced tube voltage and reduced intravenous (IV) contrast dose. We reviewed consecutive CTPAs performed between 9/1/2010 and 10/31/2010 on a 128-slice Siemens AS+ scanner using automated tube current modulation with quality reference mAs 200 and IV contrast concentration 370 mg I/ml followed by a saline flush: 99 scans at 120 kVp using 75 ml of contrast at 5 ml/s and 53 scans on patients lighter than 175 lbs at 100 kVp using 50 ml of contrast at 4 ml/s. We measured patient size (mean water-equivalent diameter) using a topogram analysis tool, signal (mean CT density) and noise (standard deviation) in the main pulmonary artery (MPA) on axial images, and calculated local CTDIvol from the kVp and mAs. Linear regression models were created for dependent variables ln(CTDIvol), signal, noise, and signal to noise ratio (SNR) as a function of independent variables size, age, gender, and kVp. After controlling for other variables, scanning at 100 kVp yielded CTDIvol reduction of 33 % (p < 0.0001), signal increase of 96 HU (p < 0.0001), and increased image noise (p < 0.0001), but without significant difference in SNR (p = 0.99). Relative to 120 kVp, 100-kVp CTPA allows simultaneous reduction of radiation exposure by 33 % and IV contrast dose by 33 % while maintaining image quality. Scanning at 100 kVp is recommended in all patients for whom the required mAs does not exceed maximum X-ray tube output.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial α-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional 1H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal α-helices separated by a less ordered region. A series of “alanine scan” peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Human Müllerian-Inhibiting Substance Promoter Contains a Functional TFII-I-Binding Initiator

Abstract Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (−6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.

Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition

Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.

Solution NMR Studies of Intact Lambda Repressor

Using a combination of two and one-dimensional NMR spectroscopy, it is shown that in the intact bacteriophage lambda repressor, the N-terminal domain assumes the same global structure as when it remains isolated. It is further shown that the N-terminal domain is only loosely attached to the C-terminal domain in the intact repressor.