Combined In-Solution Fragment Screening and Crystallographic Binding-Mode Analysis with a Two-Domain Hsp70 Construct.

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP.

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.

The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.

Reversibility of a Point Mutation Induced Domain Shift: Expanding the Conformational Space of a Sucrose Phosphorylase.

Despite their popularity as enzyme engineering targets structural information about Sucrose Phosphorylases remains scarce. We recently clarified that the Q345F variant of Bifidobacterium adolescentis Sucrose Phosphorylase is able to accept large polyphenolic substrates like resveratrol via a domain shift. Here we present a crystal structure of this variant in a conformation suitable for the accommodation of the donor substrate sucrose in excellent agreement with the wild type structure. Remarkably, this conformation does not feature the previously observed domain shift which is therefore reversible and part of a dynamic process rather than a static phenomenon. This crystallographic snapshot completes our understanding of the catalytic cycle of this useful variant and will allow for a more rational design of further generations of Sucrose Phosphorylase variants.

Switching enzyme specificity from phosphate to resveratrol glucosylation.

Here we present a point mutation-triggered domain shift which switches the acceptor preference of a sucrose phosphorylase from phosphate to a variety of large polyphenolic compounds including resveratrol and quercetin, enabling their efficient glucosylation. The variant possesses a high affinity for aromatic substrates due to newly introduced π-π- and hydrophobic interactions in the altered active site. The domain shift brings about a substantially enlarged and multifunctional active site for polyphenol glucosylation and rare disaccharide production. The crystal structure of the variant with its product resveratrol-3-α-d-glucoside allows the prediction of the substrate scope and regioselectivity of the aromatic compounds' glucosylation sites.

Exploring the Structural Space of the Galectin-1-Ligand Interaction.

Galectin-1 is a tumor-associated protein recognizing the Galß1-4GlcNAc motif of cell-surface glycoconjugates. Herein, we report the stepwise expansion of a multifunctional natural scaffold based on N-acetyllactosamine (LacNAc). We obtained a LacNAc mimetic equipped with an alkynyl function on the 3'-hydroxy group of the disaccharide facing towards a binding pocket adjacent to the carbohydrate-recognition domain. It served as an anchor motif for further expansion by the Sharpless-Huisgen-Meldal reaction, which resulted in ligands with a binding mode almost identical to that of the natural carbohydrate template. X-ray crystallography provided a structural understanding of the galectin-1-ligand interactions. The results of this study enable the development of bespoke ligands for members of the galectin target family.

Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions.

Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts.

Redesign of the Active Site of Sucrose Phosphorylase through a Clash-Induced Cascade of Loop Shifts.

Sucrose phosphorylases have been applied in the enzymatic production of glycosylated compounds for decades. However, several desirable acceptors, such as flavonoids or stilbenoids, that exhibit diverse antimicrobial, anticarcinogenic or antioxidant properties, remain poor substrates. The Q345F exchange in sucrose phosphorylase from Bifidobacterium adolescentis allows efficient glucosylation of resveratrol, (+)-catechin and (-)-epicatechin in yields of up to 97 % whereas the wild-type enzyme favours sucrose hydrolysis. Three previously undescribed products are made available. The crystal structure of the variant reveals a widened access channel with a hydrophobic aromatic surface that is likely to contribute to the improved activity towards aromatic acceptors. The generation of this channel can be explained in terms of a cascade of structural changes arising from the Q345F exchange. The observed mechanisms are likely to be relevant for the design of other tailor-made enzymes.

The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.

The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

Mutations in the SIX1/2 pathway and the DROSHA/DGCR8 miRNA microprocessor complex underlie high-risk blastemal type Wilms tumors.

Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

Structural basis of assembly chaperone- mediated snRNP formation.

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

Mutations in SNRPE, which encodes a core protein of the spliceosome, cause autosomal-dominant hypotrichosis simplex.

Hypotrichosis simplex (HS) comprises a group of hereditary isolated alopecias that are characterized by a diffuse and progressive loss of hair starting in childhood and shows a wide phenotypic variability. We mapped an autosomal-dominant form of HS to chromosome 1q31.3-1q41 in a Spanish family. By direct sequencing, we identified the heterozygous mutation c.1A>G (p.Met1?) in SNRPE that results in loss of the start codon of the transcript. We identified the same mutation in a simplex HS case from the UK and an additional mutation (c.133G>A [p.Gly45Ser]) in a simplex HS case originating from Tunisia. SNRPE encodes a core protein of U snRNPs, the key factors of the pre-mRNA processing spliceosome. The missense mutation c.133G>A leads to a glycine to serine substitution and is predicted to disrupt the structure of SNRPE. Western blot analyses of HEK293T cells expressing SNRPE c.1A>G revealed an N-terminally truncated protein, and therefore the mutation might result in use of an alternative in-frame downstream start codon. Subcellular localization of mutant SNRPE by immunofluorescence analyses as well as incorporation of mutant SNRPE proteins into U snRNPs was found to be normal, suggesting that the function of U snRNPs in splicing, rather than their biogenesis, is affected. In this report we link a core component of the spliceosome to hair loss, thus adding another specific factor in the complexity of hair growth. Furthermore, our findings extend the range of human phenotypes that are linked to the splicing machinery.

Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt.

Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing.

Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg.

Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di- or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl-lysine-containing histone tail peptide bound to the MBT repeat domain shows that the methyl-lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl-lysine-binding activity are required for repression of Hox genes.

Crystal structure of Bacillus subtilis S-adenosylmethionine:tRNA ribosyltransferase-isomerase.

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9A. The structure reveals two domains representing a 6-stranded beta-barrel and an alpha beta alpha-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.