Johannes Rudolph

Inhibiting transient protein–protein interactions: lessons from the Cdc25 protein tyrosine phosphatases

Transient protein–protein interactions have key regulatory functions in many of the cellular processes that are implicated in cancerous growth, particularly the cell cycle. Targeting these transient interactions as therapeutic targets for anticancer drug development seems like a good idea, but it is not a trivial task. This Review discusses the issues and difficulties that are encountered when considering these transient interactions as drug targets, using the example of the cell division cycle 25 (Cdc25) phosphatases and their cyclin-dependent kinase (CDK)–cyclin protein substrates.

Interface surfaces for protein-protein complexes

Protein-protein interactions, which form the basis for most cellular processes, result in the formation of protein interfaces. Believing that the local shape of proteins is crucial, we take a geometric approach and present a definition of an interface surface formed by two or more proteins as a subset of their Voronoi diagram. The definition deals with the difficult and important problem of specifying interface boundaries by invoking methods used in the alpha shape representation of molecules, the discrete flow on Delaunay simplices to define pockets and reconstruct surfaces, and the assessment of the importance of topological features. We present an algorithm to construct the surface and define a hierarchy that distinguishes core and peripheral regions. This hierarchy is shown to have correlation with hot-spots in protein-protein interactions. Finally, we study the geometric and topological properties of interface surfaces and show their high degree of contortion.

Protein-Protein Interfaces: Properties, Preferences, and Projections

Herein, we study the interfaces of a set of 146 transient protein-protein interfaces in order to better understand the principles of their interactions. We define and generate the protein interface using tools from computational geometry and topology and then apply statistical analysis to its residue composition. In addition to counting individual occurrences, we evaluate pairing preferences, both across and as neighbors on one side of an interface. Likelihood correction emphasizes novel and unexpected pairs, such as the His-Cys pair found in most complexes of serine proteases with their diverse inhibitors and the Met-Met neighbor pair found in unrelated protein interfaces. We also present a visualization of the protein interface that allows for facile identification of residue-residue contacts and other biochemical properties.

Differential Susceptibility of Yeast S and M Phase CDK Complexes to Inhibitory Tyrosine Phosphorylation

BACKGROUND Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.

COARSE AND RELIABLE GEOMETRIC ALIGNMENT FOR PROTEIN DOCKING

We present an efficient algorithm for generating a small set of coarse alignments between interacting proteins using meaningful features on their surfaces. The proteins are treated as rigid bodies, but the results are more generally useful as the produced configurations can serve as input to local improvement algorithms that allow for protein flexibility. We apply our algorithm to a diverse set of protein complexes from the Protein Data Bank, demonstrating the effectivity of our algorithm, both for bound and for unbound protein docking problems.

A fluorescence polarization assay for native protein substrates of kinases

Protein phosphorylation is the mediator of many important cellular processes of signal transduction and cell regulation. Phosphorylation often occurs on multiple sites within a single protein, whereby the results of individual phosphorylations are not well defined. This is partially due to the lack of tools for analyzing specific phosphorylation states in a quantitative manner. We have developed a high-throughput, rapid, and quantitative method for the determination of the phosphorylation status of peptides and, more importantly, native protein substrates of kinases using a competitive fluorescence-based approach. We have applied our method to measuring the phosphorylation activity of the Wee1 and Myt1 kinases. Our technique allows one to monitor the bis-phosphorylation status of the Cdk2 protein using an antibody specific for bis-phosphorylated Cdk2 and a fluorescently labeled bis-phosphorylated Cdk2 peptide. We have used this assay to screen a library of 16 general kinase inhibitors against Wee1 and Myt1 activity. None of the inhibitors inhibited Wee1, but both staurosporine and K-252a inhibited Myt1, with IC(50) values of 9.2+/-3.6 and 4.0+/-1.3 microM, respectively.

Evaluating the quality of NMR structures by local density of protons

Evaluating the quality of experimentally determined protein structural models is an essential step toward identifying potential errors and guiding further structural refinement. Herein, we report the use of proton local density as a sensitive measure to assess the quality of nuclear magnetic resonance (NMR) structures. Using 256 high‐resolution crystal structures with protons added and optimized, we show that the local density of different proton types display distinct distributions. These distributions can be characterized by statistical moments and are used to establish local density Z‐scores for evaluating both global and local packing for individual protons. Analysis of 546 crystal structures at various resolutions shows that the local density Z‐scores increase as the structural resolution decreases and correlate well with the ClashScore (Word et al. J Mol Biol 1999;285(4):1711–1733) generated by all atom contact analysis. Local density Z‐scores for NMR structures exhibit a significantly wider range of values than for X‐ray structures and demonstrate a combination of potentially problematic inflation and compression. Water‐refined NMR structures show improved packing quality. Our analysis of a high‐quality structural ensemble of ubiquitin refined against order parameters shows proton density distributions that correlate nearly perfectly with our standards derived from crystal structures, further validating our approach. We present an automated analysis and visualization tool for proton packing to evaluate the quality of NMR structures. Proteins 2006.

Autophosphorylation of Ser66 on Xenopus Myt1 is a Prerequisite for Meiotic Inactivation of Myt1

Myt1 is a dual-specificity kinase that contributes to the regulation of the cell cycle byadding inhibitory phosphates to the cyclin-dependent kinases (Cdk/cyclins). Myt1 is found to bephosphorylated and less active in M-phase compared to interphase. Although Myt1 can bephosphorylated by several different kinases in vitro, it is not well understood how Myt1 isregulated in vivo. Additionally, the interplay between phosphorylation by other kinases andautophosphorylation has not been investigated. Since phosphorylation is an important mode ofregulation for Myt1, we have investigated the properties and physiological significance of theautophosphorylation of Myt1 from Xenopus laevis (XMyt1). Using MALDI mass spectrometrywe have identified Ser66 and Ser76 as autophosphorylation sites. Autophosphorylation isimportant for the activity of XMyt1 in intact cells, as found by comparing the timing of the cellcycle in Xenopus oocytes expressing either exogenous wild type XMyt1 or itsautophosphorylation site mutants. Specifically, S66A is significantly more potent than wild typeXMyt1 at delaying entry into meiosis and concomitantly is hypophosphorylated as evident by aloss of mobility shift. However, this cannot be accounted for by a simple increase in kinaseactivity towards Cdk/cyclins in vitro. We therefore propose that Myt1 catalyzedautophosphorylation of residue S66 is a prerequisite and/or trigger for the furtherphosphorylation and inactivation of Myt1. Thus autophosphorylation of Myt1 is a novelinhibitory mechanism that adds another layer of complexity to the phosphorylation-dependentmechanism of Myt1 regulation.

Segmenting motifs in protein-protein interface surfaces

Protein-protein interactions form the basis for many intercellular events. In this paper we develop a tool for understanding the structure of these interactions. Specifically, we define a method for identifying a set of structural motifs on protein-protein interface surfaces. These motifs are secondary structures, akin to α-helices and β-sheets in protein structure; they describe how multiple residues form knob-into-hole features across the interface. These motifs are generated entirely from geometric properties and are easily annotated with additional biological data. We point to the use of these motifs in analyzing hotspot residues.

Facile variation of reagent concentrations in rapid quench enzymology

Detailed enzymological investigations are essential in elucidating the reaction pathway of an enzyme, including the identification of reaction intermediates, the quantitative determination of the rate-determining step(s), and the nature of the transition state. The information derived from these enzymological studies is especially important in the development of specific inhibitors that can find application in both research applications and as potential drugs. Many enzymological studies are performed using steady-state kinetics, as, for example, in determining relative affinities in high-throughput screens, determining the order of substrate binding and product release, and structure-activity relationships (SAR) [1]. However, steady-state kinetics are limited in that they only provide the kcat and Km parameters that are potentially complex functions of all the individual rate constants in the enzymatic reaction pathway. Therefore, transient state kinetics have been developed to elucidate both presteady-state and one-turnover reactions allowing for a more complete description of the individual reaction steps [2]. When possible, enzymologists attempt to develop enzyme assays that use an absorbance or fluorescent signal allowing for continuous measurement of product formation (or substrate depletion). The number of enzyme reactions that can be monitored directly using native substrates is extremely limited and has led to the use of artificial substrates that incorporate elements with detectable extinction coefficients or fluorescent properties or coupled assays. However, artificial substrates and coupling enzymes do not adequately address the needs of enzymology. First, full characterization of an enzyme requires work with native substrates and most native substrates do not have useful absorbance or fluorescent properties. Second, coupled assays are not useful in transient state kinetics where the lag time in observation of product formation through the coupling enzymes would mask the reaction of interest. Because of these limitations, enzymologists have for many years relied on rapid quench flow. In this technique, individual time points are collected along the reaction course and then analyzed by a suitable technique, often using radioactive labeling and separation of products from substrates. In order to perform these experiments rapidly enough to measure reaction kinetics on the millisecond time scale, a number of rapid quench apparatus have been developed. A rapid quench flow experiment generally involves three syringes. A number of manufacturers have specialized in making high-quality instruments capable of performing rapid quench flow on a microvolume basis, sufficiently small in volume to be useful for the enzymologist. These companies include Hi-Tech Scientific, Bio-Logic, and KinTek. One of the most common experiments performed by an enzymologist is the variation of substrate and/or enzyme concentration to determine dependencies of reaction rates on these two key variables. In many experiments, a wide-ranging substrate and/or enzyme concentration-dependence determination is not needed. For example, in a so-called single-turnover experiment, one may only be interested in determining whether one is working under conditions of limiting association between enzyme and substrate or limiting rate of reaction on the enzyme (i.e., actual chemistry) [2]. This can easily be determined by doubling the enzyme and Analytical Biochemistry 312 (2003) 80–83