New Aspects of the Phosphatase VHZ Revealed by a High-Resolution Structure with Vanadate and Substrate Screening

The recently discovered 150-residue human VHZ (VH1-related protein, Z member) is one of the smallest protein tyrosine phosphatases (PTPs) known and contains only the minimal structural elements common to all PTPs. We report a substrate screening analysis and a crystal structure of the VHZ complex with vanadate at 1.1 Å resolution, with a detailed structural comparison with other members of the protein tyrosine phosphatase family, including classical tyrosine-specific protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). A screen with 360 phosphorylated peptides shows VHZ efficiently catalyzes the hydrolysis of phosphotyrosine (pY)-containing peptides but exhibits no activity toward phosphoserine (pS) or phosphothreonine (pT) peptides. The new structure reveals a deep and narrow active site more typical of the classical tyrosine-specific PTPs. Despite the high degrees of structural and sequence similarity between VHZ and classical PTPs, its general acid IPD-loop is most likely conformationally rigid, in contrast to the flexible WPD counterpart of classical PTPs. VHZ also lacks substrate recognition domains and other domains typically found on classical PTPs. It is therefore proposed that VHZ is more properly classified as an atypical PTP rather than an atypical DSP, as has been suggested

Structure of frequency-interacting RNA helicase from <i>Neurospora crassa</i> reveals high flexibility in a domain critical for circadian rhythm and RNA surveillance

<div><p>The FRH (frequency-interacting RNA helicase) protein is the <i>Neurospora crassa</i> homolog of yeast Mtr4, an essential RNA helicase that plays a central role in RNA metabolism as an activator of the nuclear RNA exosome. FRH is also a required component of the circadian clock, mediating protein interactions that result in the rhythmic repression of gene expression. Here we show that FRH unwinds RNA substrates <i>in vitro</i> with a kinetic profile similar to Mtr4, indicating that while FRH has acquired additional functionality, its core helicase function remains intact. In contrast with the earlier FRH structures, a new crystal form of FRH results in an ATP binding site that is undisturbed by crystal contacts and adopts a conformation consistent with nucleotide binding and hydrolysis. Strikingly, this new FRH structure adopts an arch domain conformation that is dramatically altered from previous structures. Comparison of the existing FRH structures reveals conserved hinge points that appear to facilitate arch motion. Regions in the arch have been previously shown to mediate a variety of protein-protein interactions critical for RNA surveillance and circadian clock functions. The conformational changes highlighted in the FRH structures provide a platform for investigating the relationship between arch dynamics and Mtr4/FRH function.</p></div

Oligomeric state analysis of FRH.

<p>(A) A crystallographic symmetry mate in the FRH^Trig structure (gray) forms extensive contacts with the FRH monomer, resulting in a buried surface area of 2445 Ų and extension of a β sheet from the RecA1 domains and N-terminal regions of both symmetry-related molecules (bottom insert). Residues making direct contact with the adjacent FRH subunit are highlighted in red (top insert). (B) Full length FRH elutes as a single species with an estimated molecular weight of 122 kDa as determined by size exclusion chromatography, suggesting that FRH is a monomer in solution. Elution volumes corresponding to protein molecular mass standards are included at the top of the chromatogram. (C) Sedimentation velocity analytical ultracentrifugation analysis also indicates that FRH sediments as a single species, with a sedimentation coefficient of 3.90 ± 0.03 S, corresponding to a calculated molecular mass of 116.6 ± 1.5 kDa, consistent with a monomer. Representative A₂₈₀ absorption scans, residuals from fitting the data to a continuous c(s) distribution model, and sedimentation coefficient distribution (c(s) versus S) of purified full length FRH protein are shown.</p

FRH unwinds RNA substrates with a preference for a poly(A) 3’ single-stranded overhang.

<p>(A) A representative unwinding assay showing displacement over time of a radiolabeled single-stranded RNA from a 16-bp duplex with a 3’ single stranded overhang by full-length FRH, as observed on a non-denaturing polyacrylamide gel. The right-most lane indicates the position of a completely denatured RNA strand, heated to 95°C. (B) Poly(A) (●) and non(A) (▲) RNA unwinding rate constants (<i>k</i>_unw) plotted as a function of FRH concentration. Best fit curves to the data were calculated as described in ‘Materials and Methods’. Data presented corresponds to the average from three independent experiments; error bars represent SD.</p

Conserved hinge points in the arch domain.

<p>(A) The FRH^Trig structure is shown (left) with approximate hinge points circled. Zoomed in stereo views of each hinge point are shown on the right with FRH^Ortho structures (large cell PDB ID 4XGT, light blue; small cell PDB ID 5E02, dark blue) superimposed on the FRH^Trig structure (red). In each view, the region below the hinge point was aligned to highlight the displacement above the hinge point. (B) The FRH arch domain sequence is shown colored by sequence conservation, based on an alignment of 108 FRH/Mtr4 sequences as reported in Jackson, <i>et</i>. <i>al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196642#pone.0196642.ref020" target="_blank">20</a>] Observed secondary structure is displayed above the sequence. Identified hinge point residues are indicated by the triangle, circle and square symbols. Each hinge point contains a strictly conserved residue (dark orange) with a loop structure on the opposing strand.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Conserved Residues at the Mtr4 C‑Terminus Coordinate Helicase Activity and Exosome Interactions

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions

Conserved Residues at the Mtr4 C‑Terminus Coordinate Helicase Activity and Exosome Interactions

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions

Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution