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Biomedical subjects

Frank Sicheri

Publications and source records attributed to Frank Sicheri.

At least 19 recordsLinked to original sources
PubMed2007

Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK.

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.

Animals↗
PubMed2006

Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays.

The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be yen-[Ile/Leu/Val]-Ser/Thr-phi-phi-X- yen/Pro (where phi denotes a large hydrophobic residue, yen is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.

Amino Acid Motifs↗
PubMed2006

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases.

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.

Amino Acid Substitution↗
PubMed2006

Structural and functional analysis of Saccharomyces cerevisiae Mob1.

The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 A X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an alpha-helix denoted H0, a strand-like element denoted S0 and a short beta strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.

Amino Acid Sequence↗
PubMed2006

Sequence-specific recognition of RNA hairpins by the SAM domain of Vts1p.

The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1p epitomizes a subfamily of SAM domains conserved from yeast to humans that function as sequence-specific RNA-binding domains. Here we report the 2.0-A X-ray structure of the Vts1p SAM domain bound to a high-affinity RNA ligand. Specificity of RNA binding arises from the association of a guanosine loop base with a shallow pocket on the SAM domain and from multiple SAM domain contacts to the unique backbone structure of the loop, defined in part by a nonplanar base pair within the loop. We have validated NNF1 as an endogenous target of Vts1p among 79 transcripts that copurify with Vts1p. Bioinformatic analysis of these mRNAs demonstrates that the RNA-binding specificity of Vts1p in vivo is probably more stringent than that of the isolated SAM domain in vitro.

Base Pairing↗
PubMed2006

I Siah substrate!

Proteins are targeted to the E3 RING ubiquitin ligase Siah through a PxAxVxP degron motif. In this issue of Structure, House et al. (2006) present the structural basis by which Siah recognizes its degron with high affinity and specificity.

Animals↗
PubMed2005

The NMR and X-ray structures of the Saccharomyces cerevisiae Vts1 SAM domain define a surface for the recognition of RNA hairpins.

The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1 has a high affinity towards RNA hairpins containing a CUGGC pentaloop. We present the 1.6 Angstroms X-ray crystal structure of the Vts1 SAM domain in its unliganded state, and the NMR solution structure of this domain in its RNA-bound state. Both structures reveal a canonical five helix SAM domain flanked by additional secondary structural elements at the N and C termini. The two structures are essentially identical, implying that no major structural rearrangements occur upon RNA binding. Amide chemical shift changes map the RNA-binding site to a shallow, basic patch at the junction of helix alpha5 and the loop connecting helices alpha1 and alpha2.

Crystallography, X-Ray↗
PubMed2005

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition.

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.

Catalysis↗
PubMed2005

Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR.

In response to binding viral double-stranded RNA byproducts within a cell, the RNA-dependent protein kinase PKR phosphorylates the alpha subunit of the translation initiation factor eIF2 on a regulatory site, Ser51. This triggers the general shutdown of protein synthesis and inhibition of viral propagation. To understand the basis for substrate recognition by and the regulation of PKR, we determined X-ray crystal structures of the catalytic domain of PKR in complex with eIF2alpha. The structures reveal that eIF2alpha binds to the C-terminal catalytic lobe while catalytic-domain dimerization is mediated by the N-terminal lobe. In addition to inducing a local unfolding of the Ser51 acceptor site in eIF2alpha, its mode of binding to PKR affords the Ser51 site full access to the catalytic cleft of PKR. The generality and implications of the structural mechanisms uncovered for PKR to the larger family of four human eIF2alpha protein kinases are discussed.

Animals↗
PubMed2005

Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain.

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

Amino Acid Motifs↗
PubMed2005

Genome-wide surveys for phosphorylation-dependent substrates of SCF ubiquitin ligases.

The SCF (Skp1-Cullin-F-box) family of ubiquitin ligases target numerous substrates for ubiquitin-dependent proteolysis, including cell cycle regulators, transcription factors, and signal transducers. Substrates are recruited to an invariant core SCF complex through one of a large family of substrate-specific adapter subunits called F-box proteins, each of which binds multiple specific substrates, often in a phosphorylation-dependent manner. The identification of substrates for SCF complexes has proven difficult, especially given the requirement of often complex phosphorylation events for substrate recognition. The archetype for such interactions is the binding of the yeast F-box protein Cdc4 to its various substrates by means of multiple motifs that weakly match an optimal consensus called the Cdc4 phosphodegron (CPD), which is phosphorylated by cyclin-dependent kinases (CDKs) and possibly other kinases. Provided phosphodegron recognition motifs and/or the targeting kinases for SCF substrates are delineated, it is possible to use genome-wide methods to identify new substrates. Here we describe two methods for the systematic retrieval of SCF substrates based on membrane arrays of synthetic phosphopeptides and on genome-wide kinase substrate profiles. In the first approach, which identifies substrates with strong matches to the CPD, a search of the predicted yeast proteome with the optimal CPD motif identified approximately 1100 matches. A phosphopeptide membrane array corresponding to each of these sequences is then probed with recombinant Cdc4, thereby identifying potential substrates. In the second approach, which identifies substrates that lack strong CPD motifs, a genome-wide set of recombinant CDK substrates is phosphorylated and directly assayed for binding to Cdc4. The proteins corresponding to these hits from each approach can then be subjected to the more stringent criteria of phosphorylation-dependent binding to Cdc4, ubiquitination by SCF(Cdc4)in vitro, and Cdc4-dependent protein instability in vivo. Both methods have identified novel substrates of Cdc4 and may, in principle, be used to identify numerous new substrates of other SCF and SCF-like complexes from yeast to humans.

Amino Acid Sequence↗
PubMed2005

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha.

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.

Amino Acid Sequence↗
PubMed2003

Bone recognition mechanism of porcine osteocalcin from crystal structure.

Osteocalcin is the most abundant noncollagenous protein in bone, and its concentration in serum is closely linked to bone metabolism and serves as a biological marker for the clinical assessment of bone disease. Although its precise mechanism of action is unclear, osteocalcin influences bone mineralization, in part through its ability to bind with high affinity to the mineral component of bone, hydroxyapatite. In addition to binding to hydroxyapatite, osteocalcin functions in cell signalling and the recruitment of osteoclasts and osteoblasts, which have active roles in bone resorption and deposition, respectively. Here we present the X-ray crystal structure of porcine osteocalcin at 2.0 A resolution, which reveals a negatively charged protein surface that coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal lattice. On the basis of our findings, we propose a model of osteocalcin binding to hydroxyapatite and draw parallels with other proteins that engage crystal lattices.

Animals↗
PubMed2003

Formin leaky cap allows elongation in the presence of tight capping proteins.

Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.

Actin Cytoskeleton↗
PubMed2003

A polo match for Plk1.

New work by Elia et al. in this issue of Cell reveals the molecular basis of phosphopeptide recognition by the polo domain and the domain's dual function to promote substrate recognition by targeting the kinase to subcellular structures and to autoregulate the adjacent protein kinase catalytic domain.

Amino Acid Motifs↗
PubMed2003

Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.

Amino Acid Motifs↗
PubMed2003

The RNA-binding SAM domain of Smaug defines a new family of post-transcriptional regulators.

Anteroposterior patterning in Drosophila melanogaster is dependent on the sequence-specific RNA-binding protein Smaug, which binds to and regulates the translation of nanos (nos) mRNA. Here we demonstrate that the sterile-alpha motif (SAM) domain of Smaug functions as an RNA-recognition domain. This represents a new function for the SAM domain family, which is well characterized for mediating protein-protein interactions. Using homology modeling and site-directed mutagenesis, we have localized the RNA-binding surface of the Smaug SAM domain and have elaborated the RNA consensus sequence required for binding. Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain-containing proteins, suggesting that the function of the domain is conserved from yeast to humans. We show here that the SAM domain of Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and that Vts1 induces transcript degradation through a mechanism involving the cytoplasmic deadenylase CCR4. Together, these results suggest that Smaug and Vts1 define a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism.

Amino Acid Sequence↗