Structure determination of human Lck unique and SH3 domains by nuclear magnetic resonance spectroscopy

BACKGROUND: Protein tyrosine kinases are involved in signal transduction pathways that regulate cell growth, differentiation, activation and transformation. Human lymphocyte specific kinase (Lck) is a 56 kDa protein involved in T-cell- and IL2-receptor signaling. Three-dimensional structures are known for SH3, SH2 and kinase domains of Lck as well as for other tyrosine kinases. No structure is known for the unique domain of any Src-type tyrosine kinase. RESULTS: Lck(1–120) comprising unique and SH3 domains was structurally investigated by nuclear magnetic resonance spectroscopy. We found the unique domain, in contrast to the SH3 part, to have basically no defined structural elements. The solution structure of the SH3 part could be determined with very high precision. It does not show significant differences to Lck SH3 in the absence of the unique domain. Minor differences were observed to the X-ray structure of Lck SH3. CONCLUSION: The unique domain of Lck does not contain any defined structure elements in the absence of ligands and membranes. Presence of the unique domain is not relevant to the three-dimensional structure of the Lck SH3 domain

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli

Background: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. Methodology/Principal Findings: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. Conclusions/Significance: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts o

The non-structural protein 5A (NS5A) of hepatitis c virus interacts with the SH3 domain of human Bin1 using non-canonical binding sites

The hepatitis C virus (HCV) is a major human pathogen that causes severe diseases such as chronic hepatitis, liver cirrhosis and finally hepatocellular carcinoma. Although no enzymatic activity could be attributed yet to the HCV non-structural protein 5A (NS5A), it is indispensable for viral replication and particle assembly. Furthermore, it is associated with a variety of cellular pathways, although their relevance for viral pathogenesis still has to be elucidated. To fulfil its function NS5A interacts with a large number of different proteins including both viral and human ones. NS5A is organized into three domains, which are connected via two low complexity sequences (LCS). The first domain is highly conserved among different HCV genotypes and forms a well-defined globular structure [1]. The domains 2 (D2) and 3 (D3) are less conserved and intrinsically disordered. Nonetheless, three segments in LCS-I and D2 show significant propensities to adopt a-helical structures as could be shown by nuclear magnetic resonance (NMR) chemical shift and 15 N relaxation data [2]. The LCS-II connecting D2 and D3 contains two directly neighbored class II PxxP-motifs, which are important for interactions with Src homology 3 (SH3) domains. SH3 domains mediate protein-protein interactions, often via binding to polyproline II helices. Recent studies also revealed alternative binding mechanisms, mainly involving helical motifs and positively charged amino acid residues. The SH3 domain of the bridging integrator 1 (Bin1) is known to interact with NS5A not only via its PxxP-motifs, but also via two non-canonical binding sites, which will be further described here [3]

Immobilization of Homogeneous Monomeric, Oligomeric and Fibrillar Aβ Species for Reliable SPR Measurements

There is strong evidence that the amyloid-beta peptide (Aß) plays a central role in the pathogenesis of Alzheimer’s disease (AD). In this context, a detailed quantitative description of the interactions with different Aß species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aß in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aß conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aß allows reliable SPR measurements and quality control of the immobilized Aß aggregate species at any step throughout the experiment

The N-Terminus of Nef from HIV-1/SIV Associates with a Protein Complex Containing Lck and a Serine Kinase

AbstractThe Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV- infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic α-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity