Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The ‘U three protein’ (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis.This work was supported by Deutsche Forschungsgemeinschaft (DFG) (SFB638, Z4 to I. S. and HU363/15-1 to E.H. and the Leibniz programme to I.S.); Cluster of Excellence CellNetworks (EcTOP1 to I.S. and E.H.); Funding for open access charge: DFG [Leibniz Programme]. M.K. was funded by a Kekule Fellowship (VCI)

Regulation of Serine Proteases in Blood Clotting and Beyond

Blood clotting is a crucial step in the wound healing process. In a series of proteolytic cleavage reactions, inactive blood clotting factors (zymogens) are transformed into active enzymes. The physiologic activator of blood clotting is the tissue factor – factor VIIa (TF-FVIIa) complex. Upon vascular damage, the integral membrane protein TF is exposed and binds the serine protease FVIIa. TF-FVIIa drives blood clotting through proteolytic cleavage of its two major protein substrates, the zymogens factor IX (FIX) and factor X (FX). The activation of FIX and FX is facilitated by TF residues located adjacent to or within the putative substrate binding site on TF (the TF “exosite”). Previous studies have shown that mutating TF exosite and exosite-adjacent residues leads to a strong decrease in activation of FIX and FX. However, it remains unclear how TF-FVIIa exhibits selectivity between these substrates. We hypothesized that an exosite-adjacent TF serine loop mediates substrate selectivity by the TF-FVIIa complex. Through extensive mutagenesis studies in combination with enzymatic assays, we determined that the length of this TF serine loop affected FIX and FX activation very differently. While FX activation was decreased by up to 200-fold when the serine loop length was changed by just one residue, FIX activation was largely unaffected. The serine loop seems to regulate the TF exosite during FX activation but has no effect on the exosite during FIX activation. The results of this study suggest that the TF-FVIIa complex actively selects between its major protein substrates, which is mediated by a TF serine loop. TF residues are not just involved in TF-FVIIa substrate selectivity but also in substrate recognition. While it is known that mutating TF exosite residues leads to decreased FX activation, it is unclear how the TF exosite interacts with FX. We hypothesized that portions of the FX light chain bind to the TF exosite to facilitate substrate activation. To test this hypothesis, we generated a stable membrane-bound complex comprising TF, FVIIa and a FX mimetic (XK1). XK1 is a hybrid protein that has increased affinity for TF-FVIIa and consists of the FX light chain bound to the Kunitz 1 domain of tissue factor pathway inhibitor. The TF-FVIIa-XK1 complex was generated, validated, and then imaged using negative stain and cryo-electron microscopy (EM). Our preliminary cryo-EM model, the first model of TF-FVIIa bound to a substrate, indicates potential interactions between the TF exosite and FX light chain. These interactions could mediate substrate recognition by the TF-FVIIa complex. Serine proteases are not just important for blood clotting but are also involved in many other cellular processes. Serine proteases are typically synthesized as inactive precursors (zymogens) which remain inactive until they reach their target location. However, in some cases serine proteases can be prematurely activated, leading to severe diseases. We hypothesized that millimolar concentrations of ATP and other nucleotides in the Endoplasmic Reticulum (ER) and Golgi could keep prematurely activated zymogens enzymatically inactive while they transition through these compartments. Our kinetic, binding and structural studies revealed that serine proteases are inhibited at low millimolar concentrations of nucleotides. ADP and ATP act as uncompetitive inhibitors and bind to serine proteases cooperatively. Inhibition of serine protease activity by ATP and other nucleotides may serve as a safety mechanism to prevent cellular damage caused by premature activation of proteases.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/172603/1/fbirkle_1.pd

A serine loop in tissue factor mediates substrate selectivity by the tissue factor–factor VIIa complex

BackgroundThe tissue factor–factor VIIa (TF–FVIIa) complex is the physiologic activator of blood clotting and plays a major role in many thrombotic diseases. TF–FVIIa drives clotting through proteolytic cleavage of its major protein substrates, factor IX (FIX) and factor X (FX). However, it remains unclear how TF–FVIIa exhibits selectivity between these substrates. We previously showed that TF residues adjacent to the putative substrate binding site of TF (“exosite”) facilitate FX activation, but the role of these residues in substrate selectivity had not been tested.ObjectivesWe hypothesized that a TF serine loop (residues S160‐S163) mediates substrate selectivity by the TF–FVIIa complex.MethodsWe generated TF serine loop and exosite mutants. The mutants were tested in FIX and FX enzyme activation assays as well as thrombin generation assays.ResultsChanges in the length of the serine loop affected rates of FIX and FX activation very differently. FX activation was decreased by up to 200‐fold when the loop length was changed by just one residue. In contrast, FIX activation was largely unaffected. Substrate selectivity was also detected in thrombin generation assays. Activation assays with TF serine loop and exosite double mutants revealed that the serine loop has no effect on the exosite during FIX activation. In contrast, the serine loop regulates the exosite during FX activation.ConclusionsOur results provide new insights into how the TF‐FVIIa complex actively selects between its major protein substrates, which is mediated by a TF serine loop.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166200/1/jth15087_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166200/2/jth15087-sup-0001-FigS1-S2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166200/3/jth15087.pd

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis.

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis

Model of co-transcriptional assembly of the 90S pre-ribosome.

<p>The nascent 5'-ETS (black line) recruits the early 90S modules (UTP-A, UTP-B, and U3 snoRNP) in a hierarchical fashion, with the UTP-A complex being the first one that binds to the extreme 5'-end of the pre-rRNA. This early assembly intermediate, together with the subsequently transcribed pre-18S rRNA (yellow line) and additional factors, forms the 90S pre-ribosome. Complexes are labeled accordingly. The 3'-hinge region is highlighted in pink. Figure is adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>].</p

Uncommon Velcro-closure of the C-terminal β-propeller 2.

<p><b>(A)</b> Schematic representation of the last blade 14 of Utp4. The four β-strands of the blade are represented as arrows in different colours: 14A and B (C-terminus of β-propeller 2, red), 14C (N-terminus of β-propeller 1, blue), and 14D ((His)₆-TEV-tag, grey). <b>(B)</b> Close-up of blade 14 complemented by the very N-terminus of the polypeptide chain, forming an uncommon parallel β-strand 14C (blue) and the artificial TEV site (grey) forming an antiparallel β-strand 14D. The highly conserved residues and their hydrogen-bonding network stabilizing the blade and therefore the Velcro-closure of β-propeller 2 are represented in sticks. Salt-bridges are indicated by dashed lines.</p

Structure of Utp4 from <i>Chaetomium thermophilum</i>.

<p><b>(A)</b> Domain architecture of Utp4. Domains present in the crystal structure are given by residue numbers and are highlighted in colour. The N-terminal β-propeller 1 covers residues from 38 to 381 and is shown in blue and the C-terminal β-propeller 2 (residues 393 to 890) in red. (His)₆-tag and TEV-site are represented in grey. <b>(B)</b> The overall structure of Utp4 presents two 7-bladed β-propellers in tandem. N- and C-termini are indicated and blades are numbered. Each β-blade consists of four β-strands (ABCD). <b>(C)</b> Tertiary interaction of β-propellers. A hairpin between β-strands 2A and 2B of β-propeller 1 packs against an α-helix between β-strands 10D and 11A of β-propeller 2 (view rotated by 90° in respect to <b>A</b>). <b>(D)</b> Surface charge (left panel) and conservation (right panel) of Utp4. The electrostatic surface (red: negative, blue: positive, contoured at ±5 <i>k</i>_<i>B</i>T/e) indicates extended positively charged patches in both β-propellers. Sequence conservation mapped on the molecular surface (magenta: conserved, cyan: variable) is most pronounced around a highly positive charged patch at the N-terminus (indicated with ‘N’).</p

Utp4 in context of the 5'-ETS and 90S pre-ribosome.

<p><b>(A)</b> The crystal structure of Utp4 (rainbow colours) placed into its cryo-EM density (overall 7.3 Å resolution of the particle) in context of the 90S pre-ribosome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>]. The density (grey mesh) is contoured at a 3σ level. The Utp4 structure fits in the EM-density as rigid body validating the relative propeller orientation and loop conformations as seen in the crystal structure in the physiological context. <b>(B)</b> Utp4 (blue: propeller 1 (Utp4-N); red: propeller 2 (Utp4-C)) in context of the entire 5'-ETS <i>de novo</i> modeled (rainbow) in the cryo-EM density. RNA helices and nucleotides at special positions are given. The 5’-end is hidden in the continuous stack of RNA helices 1 and 2. Single stranded RNA-parts are indicated by connecting lines (grey). <b>(C)</b> Utp4/5'-ETS in context of a close-up of the UTP-A complex as part of the entire 90S pre-ribosome complex (grey). The region of U3 snoRNA base-paring at the 3'-end of the 5'-ETS (beyond nucleotide 243) is highlighted in cyan. <b>(D)</b> Utp4/5'-ETS/Utp8 interaction around nucleotide G66 (magenta) identified as major contact point by CRAC analysis. Left panel: Utp4 is indicated by surface potential map (±5 <i>k</i>_<i>B</i>T/e, blue positive). The C-terminus of Utp8 (Utp8-C, end of predicted α-helix and β-strand, no sequence modeled) are given in orange. The α-helix is <i>de novo</i> placed as ideal helix in the cryo-EM density, whereas the β-strand is taken from the artificial strand 14D of the X-ray structure (connection given as dashed lines is unclear). Right panel: Model for the Utp8-C interaction with Utp4. The Velcro-closed ‘2+1+1’ blade 14 is completed <i>in trans</i> by Utp8 and G66 binds to the positive patch (indicated by R³⁴⁴ and K³⁸³) in the Utp4-N/Utp4-C interface.</p

<i>In vitro</i> protein-RNA UV crosslinking analysis of <i>ct</i>Utp4.

<p><b>(A)</b> Hits obtained from deep sequencing analysis of <i>Chaetomium thermophilum</i> His₆-Utp4 (coverage, blue) mapped within the 5'-ETS (nucleotides 1 to 587) after UTP-A/5'-ETS RNP assembly by co-expression in yeast. <b>(B)</b> Mutations (deletions and substitutions) identified after cDNA library synthesis are indicated by red bars. Mutational hot spots observed in the two crosslinked regions are labeled accordingly (G66 and A220). As background control, the UTP-A/5'-ETS complex carrying untagged Utp4 (“no His₆ tag”) was used. The crosslinked region around 5'-ETS bases 100–140, which was found also in the untagged control, is marked with an asterisk. <b>(C)</b> and <b>(D)</b> The two main regions of the 5'-ETS (A53-C96 and A192-C235) that were crosslinked to His₆-Utp4 are shown together the number of mutations per base. The respective 5'-ETS sequence is depicted below. Mutational hot spots G66 and A220 colored in red.</p