The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation.

The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes.

Population shuffling of protein conformations.

Motions play a vital role in the functions of many proteins. Discrete conformational transitions to excited states, happening on timescales of hundreds of microseconds, have been extensively characterized. On the other hand, the dynamics of the ground state are widely unexplored. Newly developed high-power relaxation dispersion experiments allow the detection of motions up to a one-digit microsecond timescale. These experiments showed that side chains in the hydrophobic core as well as at protein-protein interaction surfaces of both ubiquitin and the third immunoglobulin binding domain of protein G move on the microsecond timescale. Both proteins exhibit plasticity to this microsecond motion through redistribution of the populations of their side-chain rotamers, which interconvert on the picosecond to nanosecond timescale, making it likely that this "population shuffling" process is a general mechanism.

Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings.

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment.

Structure of the RBD-PRDI fragment of the antiterminator protein GlcT.

GlcT is a transcriptional antiterminator protein that is involved in regulation of glucose metabolism in Bacillus subtilis. Antiterminator proteins bind specific RNA sequences, thus preventing the formation of overlapping terminator stem-loops. The structure of a fragment (residues 3-170) comprising the RNA-binding domain (RBD) and the first regulatory domain (PRDI) of GlcT was solved at 2.0 Šresolution with one molecule in the asymmetric unit. The two domains are connected by a helical linker. Their interface is mostly constituted by hydrophobic interactions.

TAR-RNA recognition by a novel cyclic aminoglycoside analogue.

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

Molecular cladistic markers in New World monkey phylogeny (Platyrrhini, Primates).

Transpositions of primate-specific Alu elements were applied as molecular cladistic markers in a phylogenetic analysis of South American primates. Seventy-four human and platyrrhine loci containing intronic Alu elements were PCR screened in various New World monkeys and the human outgroup to detect the presence of orthologous retrotransposons informative of New World monkey phylogeny. Six loci revealed size polymorphism in the amplification pattern, indicating a shared derived character state due to the presence of orthologous Alu elements confirmed by subsequent sequencing. Three markers corroborate (1) New World monkey monophyly and one marker supports each of the following callitrichine relationships: (2) Callithrix and Cebuella are more closely related to each other than to any other callitrichine, (3) the callitrichines form a monophyletic clade including Callimico, and (4) the next living relatives to the callitrichines are Cebus, Saimiri, and Aotus.