Jinzhong Lin

Identification of cross-linked peptides from complex samples

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein–interaction studies.

Crystal structure of human mitoNEET reveals distinct groups of iron–sulfur proteins

MitoNEET is a protein of unknown function present in the mitochondrial membrane that was recently shown to bind specifically the antidiabetic drug pioglizatone. Here, we report the crystal structure of the soluble domain (residues 32–108) of human mitoNEET at 1.8-Å resolution. The structure reveals an intertwined homodimer, and each subunit was observed to bind a [2Fe-2S] cluster. The [2Fe-2S] ligation pattern of three cysteines and one histidine differs from the known pattern of four cysteines in most cases or two cysteines and two histidines as observed in Rieske proteins. The [2Fe-2S] cluster is packed in a modular structure formed by 17 consecutive residues. The cluster-binding motif is conserved in at least seven distinct groups of proteins from bacteria, archaea, and eukaryotes, which show a consensus sequence of (hb)-C-X1-C-X2-(S/T)-X3-P-(hb)-C-D-X2-H, where hb represents a hydrophobic residue; we term this a CCCH-type [2Fe-2S] binding motif. The nine conserved residues in the motif contribute to iron ligation and structure stabilization. UV-visible absorption spectra indicated that mitoNEET can exist in oxidized and reduced states. Our study suggests an electron transfer function for mitoNEET and for other proteins containing the CCCH motif.

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Box C/D RNA protein complexes (RNPs) direct site-specific 2′-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-Å resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide–substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.

Structural organization of box C/D RNA-guided RNA methyltransferase.

Box C/D guide RNAs are abundant noncoding RNAs that primarily function to direct the 2′-O-methylation of specific nucleotides by base-pairing with substrate RNAs. In archaea, a bipartite C/D RNA assembles with L7Ae, Nop5, and the methyltransferase fibrillarin into a modification enzyme with unique substrate specificity. Here, we determined the crystal structure of an archaeal C/D RNA–protein complex (RNP) composed of all 3 core proteins and an engineered half-guide RNA at 4 Å resolution, as well as 2 protein substructures at higher resolution. The RNP structure reveals that the C-terminal domains of Nop5 in the dimeric complex provide symmetric anchoring sites for 2 L7Ae-associated kink-turn motifs of the C/D RNA. A prominent protrusion in Nop5 seems to be important for guide RNA organization and function and for discriminating the structurally related U4 snRNA. Multiple conformations of the N-terminal domain of Nop5 and its associated fibrillarin in different structures indicate the inherent flexibility of the catalytic module, suggesting that a swinging motion of the catalytic module is part of the enzyme mechanism. We also built a model of a native C/D RNP with substrate and fibrillarin in an active conformation. Our results provide insight into the overall organization and mechanism of action of C/D RNA–guided RNA methyltransferases.

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs.

Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET.

The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71−H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44−108), a soluble domain of mitoNEET containing residues 44−108. Visible−UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44−108) as a result of decomposition of the iron−sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron−sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.

An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

The structure of a complex of two ribosome synthesis factors and identification of their ribosomal binding sites provides insights into early stages of ribosome biogenesis.

Structure of Utp21 tandem WD domain provides insight into the organization of the UTPB complex involved in ribosome synthesis.

Assembly of the eukaryotic ribosome requires a large number of trans-acting proteins and small nucleolar RNAs that transiently associate with the precursor rRNA to facilitate its modification, processing and binding with ribosomal proteins. UTPB is a large evolutionarily conserved complex in the 90S small subunit processome that mediates early processing of 18S rRNA. UTPB consists of six proteins Utp1/Pwp1, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 and has abundant WD domains. Here, we determined the crystal structure of the tandem WD domain of yeast Utp21 at 2.1 Å resolution, revealing two open-clamshell-shaped β-propellers. The bottom faces of both WD domains harbor several conserved patches that potentially function as molecular binding sites. We show that residues 100–190 of Utp18 bind to the tandem WD domain of Utp21. Structural mapping of previous crosslinking data shows that the WD domains of Utp18 and Utp1 are organized on two opposite sides of the Utp21 WD domains. This study reports the first structure of a UTPB component and provides insight into the structural organization of the UTPB complex.

Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits

Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1–Utp21, Utp12–Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

Box C/D guide RNAs recognize a maximum of 10 nt of substrates.

Significance Box C/D RNAs are a large family of noncoding RNAs that guide 2′-O-methylation of RNAs. These RNAs associate with three or four proteins into C/D ribonucleoproteins (RNPs). The guide region of C/D RNAs is variable in length, particularly in eukaryotes, and by prediction, it can form 10–21 bp with substrates. Crystallographic and biochemical analyses revealed that the guide recognizes only a maximum of 10 nt in a substrate. Longer guide–substrate duplexes need to be unwound to fit into a size-limiting protein channel for modification. Our study reveals an aspect of the substrate recognition mechanism of C/D RNA. This mechanism is incompatible with the RNA-swapped model for dimeric C/D RNP. Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide–substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.