Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH.

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

Cyclin B and cyclin A confer different substrate recognition properties on CDK2.

The transitions of the cell cycle are regulated by the cyclin dependent protein kinases (CDKs). The cyclins activate their respective CDKs and confer substrate recognition properties. We report the structure of phospho-CDK2/cyclin B and show that cyclin B confers M phase-like properties on CDK2, the kinase that is usually associated with S phase. Cyclin B produces an almost identical activated conformation of CDK2 as that produced by cyclin A. There are differences between cyclin A and cyclin B at the recruitment site, which in cyclin A is used to recruit substrates containing an RXL motif. Because of sequence differences this site in cyclin B binds RXL motifs more weakly than in cyclin A. Despite similarity in kinase structures, phospho-CDK2/cyclin B phosphorylates substrates, such as nuclear lamin and a model peptide derived from p107, at sequences SPXX that differ from the canonical CDK2/cyclin A substrate recognition motif, SPXK. CDK2/cyclin B phosphorylation at these non-canonical sites is not dependent on the presence of a RXL recruitment motif. The p107 peptide contains two SP motifs each followed by a non-canonical sequence of which only one site (Ser640) is phosphorylated by pCDK2/cyclin A while two sites are phosphorylated by pCDK2/cyclin B. The second site is too close to the RXL motif to allow the cyclin A recruitment site to be effective, as previous work has shown that there must be at least 16 residues between the catalytic site serine and the RXL motif. Thus the cyclins A and B in addition to their role in promoting the activatory conformational switch in CDK2, also provide differential substrate specificity.

The role of the phospho-CDK2/cyclin A recruitment site in substrate recognition.

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 A from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the gamma-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 microm) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

Structures of the Dsk2 UBL and UBA domains and their complex.

The yeast proteins Dsk2 and Rad23 belong to a family of proteins that contain an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). Both Dsk2 and Rad23 function as adaptors to target ubiquitin-labelled proteins to the proteasome through recognition of polyubiquitin (four or more K48-linked ubiquitins) by their UBA domains and to the yeast proteasomal subunit Rpn1 by their UBL domains. The crystal structures of the Dsk2 UBL domain, the Dsk2 UBA domain and the Dsk2 UBA-UBL complex are reported. In the crystal, the Dsk2 UBA domains associate through electrostatic interactions to form ninefold helical ribbons that leave the ubiquitin-binding surface exposed. The UBA-UBL complex explains the reduced affinity of the UBA domain for UBL compared with ubiquitin and has implications for the regulation of Dsk2 adaptor function during ubiquitin-mediated proteasomal targeting. A model is discussed in which two or more Dsk2 UBA molecules may selectively bind to K48-linked polyubiquitin.

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles.

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

The crystal structure of human CDK7 and its protein recognition properties.

CDK7, a member of the cyclin-dependent protein kinase family, regulates the activities of other CDKs through phosphorylation on their activation segment and hence contributes to control of the eukaryotic cell cycle. CDK7 also assists in the regulation of transcription as part of the transcription factor TFIIH complex. For maximum activity and stability, CDK7 requires phosphorylation, association with cyclin H, and association with a third protein, MAT1. We have determined the crystal structure of human CDK7 in complex with ATP at 3 A resolution. The kinase is in the inactive conformation, similar to that observed for inactive CDK2. The activation segment is phosphorylated at Thr170 and is in a defined conformation that differs from that in phospho-CDK2 and phospho-CDK2/cyclin A. The functional properties of the enzyme against CDK2 and CTD as substrates are characterized through kinase assays. Experiments confirm that CDK7 is not a substrate for kinase-associated phosphatase.

Alternative binding modes of an inhibitor to two different kinases.

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.

Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A.

Progression through S phase of the eukaryotic cell cycle is regulated by the action of the cyclin dependent protein kinase 2 (CDK2) in association with cyclin A. CDK2/cyclin A phosphorylates numerous substrates. Substrate specificity often employs a dual recognition strategy in which the sequence flanking the phospho-acceptor site (Ser.Pro.X.Arg/Lys) is recognized by CDK2, while the cyclin A component of the complex contains a hydrophobic site that binds Arg/Lys.X.Leu ("RXL" or "KXL") substrate recruitment motifs. To determine additional sequence specificity motifs around the RXL sequence, we have performed X-ray crystallographic studies at 2.3 A resolution and isothermal calorimetry measurements on complexes of phospho-CDK2/cyclin A with a recruitment peptide derived from E2F1 and with shorter 11-mer peptides from p53, pRb, p27, E2F1, and p107. The results show that the cyclin recruitment site accommodates a second hydrophobic residue either immediately C-terminal or next adjacent to the leucine of the "RXL" motif and that this site makes important contributions to the recruitment peptide recognition. The arginine of the RXL motif contacts a glutamate, Glu220, on the cyclin. In those substrates that contain a KXL motif, no ionic interactions are observed with the lysine. The sequences N-terminal to the "RXL" motif of the individual peptides show no conservation, but nevertheless make common contacts to the cyclin through main chain interactions. Thus, the recruitment site is able to recognize diverse but conformationally constrained target sequences. The observations have implications for the further identification of physiological substrates of CDK2/cyclin A and the design of specific inhibitors.

Structural studies with inhibitors of the cell cycle regulatory kinase cyclin-dependent protein kinase 2.

Components of the cell cycle machinery are frequently altered in cancer. Many of these alterations affect the cyclin-dependent kinases (CDKs) and their regulation. Staurosporine and 7-hydroxystaurosporine (UCN-01) are two natural product kinase inhibitors originally identified as potent protein kinase C inhibitors. Staurosporine is non-selective and too toxic for use in therapy, but UCN-01 shows greater selectivity, and is in clinical trials. We have determined the crystal structures of staurosporine bound to monomeric CDK2 and UCN-01 bound to active phospho-CDK2/cyclin A. Both compounds mimic the hydrogen bonds made by the adenine moiety of ATP, and both exploit the non-polar nature of the adenine-binding site. In the complex with UCN-01, a hydrogen-bonded water molecule is incorporated into the non-polar cavity, which provides a partial polar character in the environment of the 7-hydroxyl group. Comparison of the ATP-binding site of CDK2 with that of other kinases reveals that in Chk1 kinase, a major target for UCN-01 in the cell, one of the surrounding residues, Ala144 in CDK2, is a serine in Chk1, thus providing a possible explanation for the effectiveness of UCN-01 against this kinase. For cells to exit mitosis, the CDKs must be completely inactivated, firstly by the ubiquintin-mediated destruction of the cyclins, followed by dephosphorylation of phospho-Thr160 (in CDK2) catalysed by the kinase-associated phosphatase and protein phosphatase 2C. We describe the structure of phospho-CDK2 in complex with kinase-associated phosphatase, and discuss the substrate recognition promoted by interactions that are remote from the catalytic site.