Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.LRRK2 | Parkinson's disease | structural modeling | EM | CL-MS

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metaldependent enzyme that releases products with 2 -3 cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.crystallography ͉ NendoU ͉ protein structure ͉ RNase family S mall nucleolar RNAs (snoRNAs), required for processing and modification of rRNA, are either independently transcribed or encoded in introns. The generation of most intronencoded snoRNAs relies on the splicing reaction and leads to equimolar accumulation of spliced mRNA and snoRNA (1). Some snoRNAs are, instead, produced by a splicing-independent pathway: Endonucleolytic cleavages of the pre-mRNA release a pre-snoRNA that is converted to the mature form by exonucleolytic trimming (2, 3). In yeast, Rnt1p was shown to be the endonuclease responsible for the excision of intron-encoded snoRNAs and to be activated by the interaction with snoRNP factors assembled on the nascent transcript (3). Among higher eukaryotes, the endoribonuclease from Xenopus laevis, called XendoU, was shown to be responsible for processing the intronencoded U16 and U86 snoRNAs (2, 4-6). XendoU cuts the RNA substrate at the level of short single-stranded uridine stretches. XendoU is unique among known endoribonucleases, because it generates products with 2Ј-3Ј cyclic phosphate and 5Ј OH termini and requires Mn 2ϩ as an essential cofactor. XendoU is broadly conserved among metazoans (HomoloGene: 48394), even if the function of the homologous proteins is still hypothetical, as in the case of the human homolog (hpp11), described as a putative serine protease (7). Notably, a protein with sequence similarity to XendoU has been recently characterized in ssRNA(ϩ) viruses of the Nidovirales order, including the coronavirus (CoV) responsible for the severe acute respiratory syndrome (SARS) (8). Studies aimed at characterizing the genome-proteome of SARS-CoV led to the isolation of a homolog of XendoU called NendoU. NendoU is a component of the replicase-transcriptase complex and exerts a critical role in virus replication and transcription (9). Interestingly, NendoU was found to cleave RNA at the level of uridines, releasing 2Ј-3Ј cyclic phosph...

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.

Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The x-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 A, shows the typical beta-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln(63)-Tyr(64)-Gly(65), is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The x-ray structure provides interesting clues as to how the spectroscopic properties are fine tuned by the chromophore environment.