SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation.

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet, some protein folds are easier to design than others. Previous work identified the 43-residue ɑßßɑ fold as especially challenging: The best designs had only a 2% success rate, compared to 39 to 87% success for other simple folds [G. J. Rocklin et al., Science 357, 168-175 (2017)]. This suggested the ɑßßɑ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over 10,000 new ɑßßɑ proteins and found over 3,000 of them to fold into stable structures using a high-throughput protease-based assay. NMR, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed ɑßßɑ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the ɑßßɑ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme.

Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.

Subpocket Similarity-Based Hit Identification for Challenging Targets: Application to the WDR Domain of LRRK2.

We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.

The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase.

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.

Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates.

Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1.

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

The MLL1 trimeric catalytic complex is a dynamic conformational ensemble stabilized by multiple weak interactions.

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 ß-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli.

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation.

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin are poorly defined. Here we show that the linked tandem Tudor and plant homeodomain (PHD) of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1) operates as a functional unit in cells, providing a defined combinatorial readout of a heterochromatin signature within a single histone H3 tail that is essential for UHRF1-directed epigenetic inheritance of DNA methylation. These findings provide critical support for the "histone code" hypothesis, demonstrating that multivalent histone engagement plays a key role in driving a fundamental downstream biological event in chromatin.

Identification and characterization of the first fragment hits for SETDB1 Tudor domain.

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.

Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1.

The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost.

The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013.

The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100% of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90% of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Heat-induced structural and chemical changes to a computationally designed miniprotein.

The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.

A Target Class Ligandability Evaluation of WD40 Repeat-Containing Proteins.

Target class-focused drug discovery has a strong track record in pharmaceutical research, yet public domain data indicate that many members of protein families remain unliganded. Here we present a systematic approach to scale up the discovery and characterization of small molecule ligands for the WD40 repeat (WDR) protein family. We developed a comprehensive suite of protocols for protein production, crystallography, and biophysical, biochemical, and cellular assays. A pilot hit-finding campaign using DNA-encoded chemical library selection followed by machine learning (DEL-ML) to predict ligands from virtual libraries yielded first-in-class, drug-like ligands for 7 of the 16 WDR domains screened, thus demonstrating the broader ligandability of WDRs. This study establishes a template for evaluation of protein family wide ligandability and provides an extensive resource of WDR protein biochemical and chemical tools, knowledge, and protocols to discover potential therapeutics for this highly disease-relevant, but underexplored target class.

Structural analysis of HopPmaL reveals the presence of a second adaptor domain common to the HopAB family of Pseudomonas syringae type III effectors.

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen Pseudomonas syringae. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308-385. While structurally similar, these domains do not share significant sequence similarity and most importantly demonstrate significant differences in key residues involved in host protein recognition, suggesting that each of them targets a different host protein.

Prediction and Validation of a Protein's Free Energy Surface Using Hydrogen Exchange and (Importantly) Its Denaturant Dependence.

The denaturant dependence of hydrogen-deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual H-bonds and map the free energy surface (FES) of a protein including the very rare states. Molecular dynamics (MD) can identify each partial unfolding event with atomic-level resolution. Hence, their combination provides a great opportunity to test the accuracy of simulations and to verify the interpretation of HDX data. For this comparison, we use Upside, our new and extremely fast MD package that is capable of folding proteins with an accuracy comparable to that of all-atom methods. The FESs of two naturally occurring and two designed proteins are so generated and compared to our NMR/HDX data. We find that Upside's accuracy is considerably improved upon modifying the energy function using a new machine-learning procedure that trains for proper protein behavior including realistic denatured states in addition to stable native states. The resulting increase in cooperativity is critical for replicating the HDX data and protein stability, indicating that we have properly encoded the underlying physiochemical interactions into an MD package. We did observe some mismatch, however, underscoring the ongoing challenges faced by simulations in calculating accurate FESs. Nevertheless, our ensembles can identify the properties of the fluctuations that lead to HDX, whether they be small-, medium-, or large-scale openings, and can speak to the breadth of the native ensemble that has been a matter of debate.

Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease.

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.

De novo design of immunoglobulin-like domains.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.