Expression, purification and crystallization of cysteine-rich human protein muskelin in Escherichia coli.

The increasing interest in the structural arrangements and functional interdependencies of individual modules within large multidomain proteins requires the development of new methods allowing efficient production and purification of large human proteins. Heterologous expression in bacteria is still the most convenient and widely-used approach. However, most of the existing tools are not well suited to expression of cysteine-rich proteins in a native-like soluble form, and with the increasing protein size refolding may result in obtaining non-native conformations or improper disulfide bridging pattern. Here, we present an efficient method of expression and purification of muskelin, a large, multidomain, cysteine-rich eukaryotic protein involved in cell adhesion and regulation of cytoskeleton dynamics. Using a broad range of purification and solubility tags, expression strains and conditions we optimized the procedure to acquire a natively folded protein of crystallization-scale quantity and purity. The correct protein conformation and disulfide bonding were anticipated from the results of circular dichroism spectra and Ellman's assay. Successful crystallization trials are a step towards muskelin crystal-structure determination, while the optimized expression and purification procedure can easily be applied to produce other eukaryotic proteins in the bacterial expression system.

Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG.

PDZ domains are ubiquitous protein-protein interaction modules which bind short, usually carboxyterminal fragments of receptors, other integral or membrane-associated proteins, and occasionally cytosolic proteins. Their role in organizing multiprotein complexes at the cellular membrane is crucial for many signaling pathways, but the rules defining their binding specificity are still poorly understood and do not readily explain the observed diversity of their known binding partners. Two homologous RhoA-specific, multidomain nucleotide exchange factors PDZRhoGEF and LARG contain PDZ domains which show a particularly broad recognition profile, as suggested by the identification of five diverse biological targets. To investigate the molecular roots of this phenomenon, we constructed a phage display library of random carboxyterminal hexapeptides. Peptide variants corresponding to the sequences identified in library selection were synthesized and their affinities for both PDZ domains were measured and compared with those of peptides derived from sequences of natural partners. Based on the analysis of the binding sequences identified for PDZRhoGEF, we propose a sequence for an 'optimal' binding partner. Our results support the hypothesis that PDZ-peptide interactions may be best understood when one considers the sum of entropic and dynamic effects for each peptide as a whole entity, rather than preferences for specific residues at a given position.

PDZ domains - common players in the cell signaling.

PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.

PDZ domain from Dishevelled -- a specificity study.

Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt ß-catenin pathway at the expense of non-canonical Wnt signaling.