Radu Huculeci

Structural insights into the intertwined dimer of fyn SH2

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide‐bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2s dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand.

Purification, crystallization and preliminary X-ray diffraction analysis of the Fyn SH2 domain and its complex with a phosphotyrosine peptide.

SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide.

1H, 13C and 15N backbone and side-chain chemical shift assignment of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

SH2 domains are interaction modules uniquely dedicated to recognize phosphotyrosine sites, playing a central role in for instance the activation of tyrosine kinases or phosphatases. Here we report the 1H, 15N and 13C backbone and side-chain chemical shift assignments of the SH2 domain of the human protein tyrosine kinase Fyn, both in its free state and bound to a high-affinity phosphotyrosine peptide corresponding to a specific sequence in the hamster middle-T antigen. The BMRB accession numbers are 17,368 and 17,369, respectively.

Dynamically Coupled Residues within the SH2 Domain of FYN Are Key to Unlocking Its Activity

Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family.

Strategic and Innovation Networks in the Flanders Biotechnology Industry

For organizations in high-technology industries, knowledge is a critical resource that can be accessed through inter-organizational networks. However, for industries characterized by a heterogeneous set of actors, little is known about how different networks within the industry interact. Therefore, our research question is: How similar are the strategic network and the innovation network in the biotechnology industry? To answer our research question, we study two networks of interest. First, the Board-of-Directors-network serves as a proxy for the strategic network that fosters knowledge transfer between organizations. Second, we analyze the innovation network by using the patent network that emerged from collaborative innovation activities. Subject of analysis is the Flanders biotechnology industry, which is characterized by strong performing research institutions, large firms and innovative SMEs. We use social network analysis methods to measure the similarity of both networks and to identify their key actors. We find that a connection between two organizations in the strategic network increases the probability of forming a new connection between the same organizations in the innovation network, or vice versa. This shows that collaborations between two organizations on one network level can lead to an interlocking of the organizations at other network levels. Our results also suggest that few companies establish and maintain a strong position in the biotechnology innovation network. This network is dominated by academic institutions, which are the key producers of scientific knowledge. Interestingly, the BoD-network has a more balanced composition and power structure and knowledge on strategic issues is transferred across a wide range of industrial actors. We also highlight the strong position of spin-off companies in the BoD-network and the absence of large firms in both networks. Our findings call for more research on the causal mechanisms of network formation and on the relationship between multiple networks within one industry.

1 H, 13 C, and 15 N backbone and side-chain chemical shift assignments of the free and bound forms of the human PTPN11 second SH2 domain

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the 1H, 15N and 13C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.

Structural Characterization of Monomeric/Dimeric State of p59(fyn) SH2 Domain.

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59fyn SH2 domain.