Promoting Substrate Binding Research Articles

U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing

To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5' splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5' splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3' splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5' splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations--a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.

The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding.

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex.

The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.

Kinetic Analysis of Arabidopsis Phospholipase Dδ: SUBSTRATE PREFERENCE AND MECHANISM OF ACTIVATION BY Ca2+ AND PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.

Molecular Rulers: An Assessment of Distance and Spatial Relationships of Src Tyrosine Kinase SH2 and Active Site Regions

The three-dimensional structures of the inactive conformations of Hck and Src, members of the Src protein-tyrosine kinase family, have recently been described. In both cases, the catalytic domain lies on the opposite face of the enzyme from the SH2 and SH3 domains. The active conformation of these enzymes has not yet been described. Given the known role of the SH2 and SH3 domains in promoting substrate binding, enzyme activation likely reorients the relative spatial arrangement between the SH2/SH3 domains and the active site region. We describe herein a series of "molecular rulers" and their use in assessing the topological and spatial relationships of the SH2 and active site regions of the Src protein-tyrosine kinase. These synthetic compounds contain sequences that are active site-directed (-Glu-Glu-Ile-Ile-(F(5))Phe-, where (F(5))Phe is pentafluorophenylalanine) and SH2-directed (-Tyr(P)-Glu-Glu-Ile-Glu-), separated by a sequence of variable length. The most potent bivalent compound, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-(GABA)(3)-Tyr(P)-Glu-Glu-Ile-Glu-amide (where GABA is gamma-aminobutyric acid), displays a >120-fold enhancement in inhibitory potency relative to the simple monovalent active site-directed species, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-amide. The short linker length (3 GABA residues) between the active site- and SH2-directed peptide fragments suggests that the corresponding domains on the Src kinase can assume a nearly contiguous spatial arrangement in the active form of the enzyme.

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C.

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.