Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

Deciphering cellular and molecular determinants of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity.

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.

Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs.

Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.

Optimizing the First TPR Domain of the Human SPAG1 Protein Provides Insight into the HSP70 and HSP90 Binding Properties.

Tetratricopeptide repeat domains, or TPR domains, are protein domains that mediate protein:protein interaction. As they allow contacts between proteins, they are of particular interest in transient steps of the assembly process of macromolecular complexes, such as the ribosome or the dynein arms. In this study, we focused on the first TPR domain of the human SPAG1 protein. SPAG1 is a multidomain protein that is important for ciliogenesis whose known mutations are linked to primary ciliary dyskinesia syndrome. It can interact with the chaperones RUVBL1/2, HSP70, and HSP90. Using protein sequence optimization in combination with structural and biophysical approaches, we analyzed, with atomistic precision, how the C-terminal tails of HSPs bind a variant form of SPAG1-TPR1 that mimics the wild-type domain. We discuss our results with regard to other complex three-dimensional structures with the aim of highlighting the motifs in the TPR sequences that could drive the positioning of the HSP peptides. These data could be important for the druggability of TPR regulators.

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis.

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.

Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate.

Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13C and 15N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway.

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA.

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

Binding properties of the quaternary assembly protein SPAG1.

In cells, many constituents are able to assemble resulting in large macromolecular machineries possessing very specific biological and physiological functions, e.g. ribosome, spliceosome and proteasome. Assembly of such entities is commonly mediated by transient protein factors. SPAG1 is a multidomain protein, known to participate in the assembly of both the inner and outer dynein arms. These arms are required for the function of sensitive and motile cells. Together with RUVBL1, RUVBL2 and PIH1D2, SPAG1 is a key element of R2SP, a protein complex assisting the quaternary assembly of specific protein clients in a tissue-specific manner and associating with heat shock proteins (HSPs) and regulators. In this study, we have investigated the role of TPR domains of SPAG1 in the recruitment of HSP chaperones by combining biochemical assays, ITC, NMR spectroscopy and molecular dynamics (MD) simulations. First, we propose that only two, out of the three TPR domains, are able to recruit the protein chaperones HSP70 and HSP90. We then focused on one of these TPR domains and elucidated its 3D structure using NMR spectroscopy. Relying on an NMR-driven docking approach and MD simulations, we deciphered its binding interface with the C-terminal tails of both HSP70 and HSP90. Finally, we addressed the biological function of SPAG1 and specifically demonstrated that a SPAG1 sub-fragment, containing a putative P-loop motif, cannot efficiently bind and hydrolyze GTP in vitro Our data challenge the interpretation of SPAG1 possessing GTPase activity. We propose instead that SPAG1 regulates nucleotide hydrolysis activity of the HSP and RUVBL1/2 partners.

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution.

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3' side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5' side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2'-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U•U and G•A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

NMR assignment and solution structure of the external DII domain of the yeast Rvb2 protein.

We report the nearly complete 1H, 15N and 13C resonance assignment and the solution structure of the external DII domain of the yeast Rvb2 protein, a member of the AAA+ATPase superfamily.

Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly.

The U3 box C/D snoRNA is one key element of 90S pre-ribosome. It contains a 5΄ domain pairing with pre-rRNA and the U3B/C and U3C΄/D motifs for U3 packaging into a unique small nucleolar ribonucleoprotein particle (snoRNP). The RNA-binding protein Snu13/SNU13 nucleates on U3B/C the assembly of box C/D proteins Nop1p/FBL and Nop56p/NOP56, and the U3-specific protein Rrp9p/U3-55K. Snu13p/SNU13 has a much lower affinity for U3C΄/D but nevertheless forms on this motif an RNP with box C/D proteins Nop1p/FBL and Nop58p/NOP58. In this study, we characterized the influence of the RNP assembly protein Rsa1 in the early steps of U3 snoRNP biogenesis in yeast and we propose a refined model of U3 snoRNP biogenesis. While recombinant Snu13p enhances the binding of Rrp9p to U3B/C, we observed that Rsa1p has no effect on this activity but forms with Snu13p and Rrp9p a U3B/C pre-RNP. In contrast, we found that Rsa1p enhances Snu13p binding on U3C΄/D. RNA footprinting experiments indicate that this positive effect most likely occurs by direct contacts of Rsa1p with the U3 snoRNA 5΄ domain. In light of the recent U3 snoRNP cryo-EM structures, our data suggest that Rsa1p has a dual role by also preventing formation of a pre-mature functional U3 RNP.

Structural Features of the Box C/D snoRNP Pre-assembly Process Are Conserved through Species.

Box C/D small nucleolar ribonucleoparticles (snoRNPs) support 2'-O-methylation of several target RNAs. They share a common set of four core proteins (SNU13, NOP58, NOP56, and FBL) that are assembled on different guide small nucleolar RNAs. Assembly of these entities involves additional protein factors that are absent in the mature active particle. In this context, the platform protein NUFIP1/Rsa1 establishes direct and simultaneous contacts with core proteins and with the components of the assembly machinery. Here, we solve the nuclear magnetic resonance (NMR) structure of a complex resulting from interaction between protein fragments of human NUFIP1 and its cofactor ZNHIT3, and emphasize their imbrication. Using yeast two-hybrid and complementation assays, protein co-expression, isothermal titration calorimetry, and NMR, we demonstrate that yeast and human complexes involving NUFIP1/Rsa1p, ZNHIT3/Hit1p, and SNU13/Snu13p share strong structural similarities, suggesting that the initial steps of the box C/D snoRNP assembly process are conserved among species.

Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis, molecular docking study and in vitro biological activities.

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A165 binding to NRP-1 (IC50=39µM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

Zf­HIT family members share the zf­HIT domain (ZHD), which is characterized by a fold in "treble-clef" through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf­HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf­HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf­HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.