Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria.

Mitochondrial polycistronic transcripts are extensively processed to give rise to functional mRNAs, rRNAs and tRNAs; starting with the release of tRNA elements through 5'-processing by RNase P (MRPP1/2/3-complex) and 3'-processing by RNase Z (ELAC2). Here, we show using in vitro experiments that MRPP1/2 is not only a component of the mitochondrial RNase P but that it retains the tRNA product from the 5'-processing step and significantly enhances the efficiency of ELAC2-catalyzed 3'-processing for 17 of the 22 tRNAs encoded in the human mitochondrial genome. Furthermore, MRPP1/2 retains the tRNA product after ELAC2 processing and presents the nascent tRNA to the mitochondrial CCA-adding enzyme. Thus, in addition to being an essential component of the RNase P reaction, MRPP1/2 serves as a processing platform for several down-stream tRNA maturation steps in human mitochondria. These findings are of fundamental importance for our molecular understanding of disease-related mutations in MRPP1/2, ELAC2 and mitochondrial tRNA genes.

SnoN Stabilizes the SMAD3/SMAD4 Protein Complex.

TGF-ß signaling regulates cellular processes such as proliferation, differentiation and apoptosis through activation of SMAD transcription factors that are in turn modulated by members of the Ski-SnoN family. In this process, Ski has been shown to negatively modulate TGF-ß signaling by disrupting active R-SMAD/Co-SMAD heteromers. Here, we show that the related regulator SnoN forms a stable complex with the R-SMAD (SMAD3) and the Co-SMAD (SMAD4). To rationalize this stabilization at the molecular level, we determined the crystal structure of a complex between the SAND domain of SnoN and the MH2-domain of SMAD4. This structure shows a binding mode that is compatible with simultaneous coordination of R-SMADs. Our results show that SnoN, and SMAD heteromers can form a joint structural core for the binding of other transcription modulators. The results are of fundamental importance for our understanding of the molecular mechanisms behind the modulation of TGF-ß signaling.

Structure of mitochondrial poly(A) RNA polymerase reveals the structural basis for dimerization, ATP selectivity and the SPAX4 disease phenotype.

Polyadenylation, performed by poly(A) polymerases (PAPs), is a ubiquitous post-transcriptional modification that plays key roles in multiple aspects of RNA metabolism. Although cytoplasmic and nuclear PAPs have been studied extensively, the mechanism by which mitochondrial PAP (mtPAP) selects adenosine triphosphate over other nucleotides is unknown. Furthermore, mtPAP is unique because it acts as a dimer. However, mtPAP's dimerization requirement remains enigmatic. Here, we show the structural basis for mtPAP's nucleotide selectivity, dimerization and catalysis. Our structures reveal an intricate dimerization interface that features an RNA-recognition module formed through strand complementation. Further, we propose the structural basis for the N478D mutation that drastically reduces the length of poly(A) tails on mitochondrial mRNAs in patients with spastic ataxia 4 (SPAX4), a severe and progressive neurodegenerative disease.

Structural basis of tumor suppressor in lung cancer 1 (TSLC1) binding to differentially expressed in adenocarcinoma of the lung (DAL-1/4.1B).

Perturbed cell adhesion mechanisms are crucial for tumor invasion and metastasis. A cell adhesion protein, TSLC1 (tumor suppressor in lung cancer 1), is inactivated in a majority of metastatic cancers. DAL-1 (differentially expressed in adenocarcinoma of the lung protein), another tumor suppressor, binds through its FERM domain to the TSLC1 C-terminal, 4.1 glycophorin C-like, cytoplasmic domain. However, the molecular basis for this interaction is unknown. Here, we describe the crystal structure of a complex between the DAL-1 FERM domain and a portion of the TSLC1 cytoplasmic domain. DAL-1 binds to TSLC1 through conserved residues in a well defined hydrophobic pocket in the structural C-lobe of the DAL-1 FERM domain. From the crystal structure, it is apparent that Tyr(406) and Thr(408) in the TSLC1 cytoplasmic domain form the most important interactions with DAL-1, and this was also confirmed by surface plasmon resonance studies. Our results refute earlier exon deletion experiments that indicated that glycophorin C interacts with the α-lobe of 4.1 FERM domains.

Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site.

The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain.

Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.

The closed structure of presequence protease PreP forms a unique 10,000 Angstroms3 chamber for proteolysis.

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase.

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.