Biomedical subjects

Markus A Seeliger

Publications and source records attributed to Markus A Seeliger.

8 recordsLinked to original sources

PubMed2006

Activation of ubiquitin ligase SCF(Skp2) by Cks1: insights from hydrogen exchange mass spectrometry.

Skp2 is the substrate recognition subunit of the multi-subunit ubiquitin ligase SCF(Skp2). It consists of an N-terminal F-box domain that binds to the Skp1 subunit and thereby tethers it to the SCF catalytic core, and an elongated C-terminal domain comprising ten Leucine-rich repeats (LRR) that binds the substrate. A small accessory protein, Cks1, is required for SCF(Skp2) to target certain substrates, including the Cyclin-dependent kinase inhibitor p27. Here we have used hydrogen/deuterium exchange monitored by mass spectrometry to investigate the mode of action of Cks1 on SCF(Skp2). We show that complex formation between Cks1 and Skp2 causes conformational changes in both proteins in regions distant from the respective binding sites. We find that Skp2 interacts with a localised region of Cks1 but the interaction causes a global change in the hydrogen exchange behaviour of Cks1. Also, whilst Cks1 binds to the most C-terminal LRRs of the elongated Skp2 molecule, the interaction induces conformational changes at the distant N-terminal LRRs, close to the F-box motif. Further, binding of Cks1 to Skp2 significantly stabilises the interaction between Skp2 and Skp1. The results reveal that the C-terminal substrate recognition region of Skp2 is coupled to the N-terminal Skp1-binding region and thereby to the SCF catalytic core; this result adds to the model proposed previously that, whilst the principal function of the F-box protein is to recruit the substrate, an additional function may be to help position the substrate in an optimal way within the SCF complex to enable efficient ubiquitin transfer.

CDC2-CDC28 Kinases↗

PubMed2006

Folding and fibril formation of the cell cycle protein Cks1.

The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine-rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases.

Adaptor Proteins, Signal Transducing↗

PubMed2005

High yield bacterial expression of active c-Abl and c-Src tyrosine kinases.

The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.

Animals↗

PubMed2005

Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins.

Cks proteins are adapter molecules that coordinate the assembly of multiprotein complexes. They share the ability to domain swap by exchanging a beta-strand, beta4. Here we use NMR spectroscopy and molecular dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCF(Skp2) ubiquitin ligation machinery. In the NMR experiment with the free form of Cks1, a subset of residues displayed elevated R2 values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale. Strikingly the region of greatest conformational variability was the beta4-strand that domain swaps to form the dimer. Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity. This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior. Short time (<5 ns) molecular dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex. A cooperative interaction between Cdk2 and Cks1 is suggested, which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding. Indications of an unusual dynamic behavior of strand beta4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations. A spontaneous reversible unzipping of hydrogen bonds between beta4 and beta2 was observed, suggesting an early intermediate structure for unfolding and/or domain swapping. We propose that the dynamic properties of the beta-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.

Allosteric Site↗

PubMed2005

Mechanism of CDK5/p25 binding by CDK inhibitors.

The cyclin-dependent kinases (CDK) CDK1, CDK2, CDK4, and CDK6 are serine/threonine protein kinases targeted in cancer therapy due to their role in cell cycle progression. The postmitotic CDK5 is involved in biological pathways important for neuronal migration and differentiation. CDK5 represents an attractive pharmacological target as its deregulation is implicated in various neurodegenerative diseases such as Alzheimer's, Parkinson's, and Niemann-Pick type C diseases, ischemia, and amyotrophic lateral sclerosis. We have generated an improved crystal form of CDK5 in complex with p25, a segment of the p35 neuronal activator. The crystals were used to solve the structure of CDK5/p25 with (R)-roscovitine and aloisine at a resolution of 2.2 and 2.3 A, respectively. The structure of CDK5/p25/roscovitine provides a rationale for the preference of CDK5 for the R over the S stereoisomer. Furthermore, roscovitine stabilized an unusual collapsed conformation of the glycine-rich loop, an important site of CDK regulation, and we report an investigation of the effects of glycine-rich loop phosphorylation on roscovitine binding. The CDK5/p25 crystals represent a valuable new tool for the identification and optimization of selective CDK inhibitors.

Crystallography, X-Ray↗

PubMed2003

Cooperative organization in a macromolecular complex.

The mechanism of assembly of multiprotein complexes and the subsequent organization of activity are not well understood. Here we report the application of biophysical tools to investigate the relationship between structure and function in protein assemblies. We used as a model system the SCF(Skp2) complex that targets p27(Kip1) for ubiquitination and subsequent degradation; this process requires an adapter protein, Cks1. By dissecting the interactions between the different subunits we show that the properties of Cks1 are highly context dependent, and its activity is acquired only when the complex is fully assembled. The results provide insights into the central role of small adapters in macromolecular assembly and explain their high sequence conservation. Simultaneous and synergistic binding of multiple subunits in a complex provides the specificity and control required before the key cell-cycle regulator p27 is committed to degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Three different binding sites of Cks1 are required for p27-ubiquitin ligation.

Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2.

The two human proteins ckshs1 and ckshs2 are each 79 amino acids in length and consist of a four-stranded beta-sheet capped at one end by two alpha-helices. They are members of the cks family of essential cell cycle regulatory proteins that can adopt two native states, a monomer and a domain-swapped dimer formed by exchange of a C-terminal beta-strand. ckshs1 and ckshs2 both have marginal thermodynamic stability (the free energies of unfolding at 25 degrees C are 3.0 and 2.5 kcal/mol, respectively) and low kinetic stability (the rates of unfolding in water are approximately 1 s(-1)). Refolding of their denatured states to the monomeric forms of the proteins is slowed by transient oligomerization that is likely to occur via domain swapping. The folding behavior of ckshs1 and ckshs2 is markedly different from that of suc1, the cks protein from Schizosaccharomyces pombe, but the domain swapping propensities are similar. The greater thermodynamic and kinetic stability of suc1 and the population of a folding intermediate are most likely a consequence of its larger size (113 residues). The similarity in the domain swapping propensities, despite the contrast in other biophysical properties, may be attributable to the common double-proline motif in the hinge loop that connects the swapped domain to the rest of the protein. The motif was shown previously for suc1 to control the equilibrium between the monomer and the domain-swapped dimer. Finally, according to our model, the kinetic barrier separating the monomer and the domain-swapped dimer arises because the protein must unfold for beta-strand exchange to occur. Consistent with this, interconversion between the two states is much faster in the human proteins than it is for suc1, reflecting the faster unfolding rates of the former.

CDC2-CDC28 Kinases↗