Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28.

The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5'-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Šresolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

Phosphorylation drives a dynamic switch in serine/arginine-rich proteins.

Serine/arginine-rich (SR) proteins are important players in RNA metabolism and are extensively phosphorylated at serine residues in RS repeats. Here, we show that phosphorylation switches the RS domain of the serine/arginine-rich splicing factor 1 from a fully disordered state to a partially rigidified arch-like structure. Nuclear magnetic resonance spectroscopy in combination with molecular dynamics simulations revealed that the conformational switch is restricted to RS repeats, critically depends on the phosphate charge state and strongly decreases the conformational entropy of RS domains. The dynamic switch also occurs in the 100 kDa SR-related protein hPrp28, for which phosphorylation at the RS repeat is required for spliceosome assembly. Thus, a phosphorylation-induced dynamic switch is common to the class of serine/arginine-rich proteins and provides a molecular basis for the functional redundancy of serine/arginine-rich proteins and the profound influence of RS domain phosphorylation on protein-protein and protein-RNA interactions.

Site-specific modification of ED-B-targeting antibody using intein-fusion technology.

BACKGROUND: A promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety. RESULTS: A full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE) attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains. CONCLUSION: Full-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.

In vitro sortagging of an antibody fab fragment: overcoming unproductive reactions of sortase with water and lysine side chains.

Sortase A from Staphylococcus aureus attracts growing interest for its use in biotechnological protein modification. This enzyme binds to a short signal sequence at the C terminus of a target protein, cleaves it by formation of an acyl-enzyme intermediate, and subsequently attaches an oligoglycine with a peptide bond. In this work, we explored its usability for the modification of the L19 Fab fragment (specific for fibronectin ED-B), a promising candidate for antibody-based cancer therapy. The Fab fragment was expressed with a sortase signal sequence attached to its light chain, and was successfully modified with a fluorescent oligoglycine probe in good yield. Our interest focused on performance under conditions of limited oligoglycine concentrations. Two unproductive side reactions of sortase were observed. The first was hydrolysis of the acyl-enzyme intermediate; in the second, sortase accepted the ε-amino group of lysine as substrate, thereby resulting in polypeptide crosslinking. In case of the L19 Fab fragment, it led to the covalent connection of the heavy and light chains. Both side reactions were effectively suppressed by sufficient concentrations of the oligoglycine probe.

Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome.

Several protein kinases, including SRPK1 and SRPK2, have been implicated in spliceosome assembly and catalytic activation. However, little is known about their targets. Here we show that SRPK1 is predominantly associated with U1 small nuclear ribonucleoprotein (snRNP), whereas SRPK2 associates with the U4/U6-U5 tri-snRNP. RNAi-mediated depletion in HeLa cells showed that SRPK2 is essential for cell viability, and it is required for spliceosomal B complex formation. SRPK2 knock down results in hypophosphorylation of the arginine-serine (RS) domain-containing human PRP28 protein (PRP28, also known as DDX23), and destabilizes PRP28 association with the tri-snRNP. Immunodepletion of PRP28 from HeLa cell nuclear extract and complementation studies revealed that PRP28 phosphorylation is required for its stable association with the tri-snRNP and for tri-snRNP integration into the B complex. Our results demonstrate a role for SRPK2 in splicing and reveal a previously unknown function for PRP28 in spliceosome assembly.