Biomedical subjects

Raimond B Ravelli

Publications and source records attributed to Raimond B Ravelli.

5 recordsLinked to original sources

PubMed2006

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1.

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.

Amino Acid Sequence↗

PubMed2006

Structural basis for budding by the ESCRT-III factor CHMP3.

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.

Amino Acid Sequence↗

PubMed2003

Characterization of photocycle intermediates in crystalline photoactive yellow protein.

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.

Bacterial Proteins↗

PubMed2002

Crystallization and preliminary X-ray analysis of the Escherichia coli adaptor protein ClpS, free and in complex with the N-terminal domain of ClpA.

Protein degradation in Escherichia coli is accomplished by a handful of large oligomeric complexes. In most cases, these proteolytic machines are comprised of a chaperone (e.g. ClpA) that is required to prepare the substrate for degradation by the peptidase (e.g. ClpP). Recently, it was shown that the substrate recognition of the chaperone ClpA could be modified by the adaptor protein ClpS. To investigate the structural implications of this change in substrate specificity, ClpS was crystallized alone and in complex with the N-terminal domain of ClpA (ClpA(N)). Crystals of ClpS diffract to 2.9 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.63, b = 145.67, c = 152.31 A. Two different crystal forms of the ClpA(N)-ClpS complex were characterized. Crystal form I (CFI) belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 91.63, b = 112.47, c = 38.47 A; data to 1.92 A resolution were collected. Crystals of form II (CFII) belong to space group P4(1/3)2(1)2, with unit-cell parameters a = b = 93.57, c = 78.77 A, and diffract to 1.85 A resolution. Data sets collected from heavy-atom derivatives of CFI indicated the incorporation of Pt and Hg atoms. Structure solution using MIR and MAD methods is currently under way.

Adenosine Triphosphatases↗

PubMed2002

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA.

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.

Adenosine Triphosphatases↗