The Core Complex of Yeast COMPASS and Human Mixed-Lineage Leukemia (MLL), Structure, Function, and Recognition of the Nucleosome.

Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

Managing macromolecular crystallographic data with a laboratory information management system.

Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example.

A snapshot love story: what serial crystallography has done and will do for us.

Serial crystallography, born from groundbreaking experiments at the Linac Coherent Light Source in 2009, has evolved into a pivotal technique in structural biology. Initially pioneered at X-ray free-electron laser facilities, it has now expanded to synchrotron-radiation facilities globally, with dedicated experimental stations enhancing its accessibility. This review gives an overview of current developments in serial crystallography, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings.

Crystal structure of the class A extended-spectrum ß-lactamase CTX-M-96 in complex with relebactam at 1.03 Angstrom resolution.

The use of ß-lactam/ß-lactamase inhibitors constitutes an important strategy to counteract ß-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A ß-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other ß-lactamases.

Preparing research samples for safe arrival at centers and facilities: recipes for successful experiments.

Preparation of biomacromolecules for structural biology studies is a complex and time-consuming process. The goal is to produce a highly concentrated, highly pure product that is often shipped to large facilities with tools to prepare the samples for crystallization trials or for measurements at synchrotrons and cryoEM centers. The aim of this article is to provide guidance and to discuss general considerations for shipping biomacromolecular samples. Details are also provided about shipping samples for specific experiment types, including solution- and cryogenic-based techniques. These guidelines are provided with the hope that the time and energy invested in sample preparation is not lost due to shipping logistics.

RNA thermometers are widespread upstream of ABC transporter genes in bacteria.

RNA thermometers are temperature-sensing non-coding RNAs that regulate the expression of downstream genes. A well-characterized RNA thermometer motif discovered in bacteria is the ROSE-like element (repression of heat shock gene expression). ATP-binding cassette (ABC) transporters are a superfamily of transmembrane proteins that harness ATP hydrolysis to facilitate the export and import of substrates across cellular membranes. Through structure-guided bioinformatics, we discovered that ROSE-like RNA thermometers are widespread upstream of ABC transporter genes in bacteria. X-ray crystallography, biochemistry, and cellular assays indicate that these RNA thermometers are functional regulatory elements. This study expands the known biological role of RNA thermometers to these key membrane transporters.

In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Šresolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.

Structural basis of nucleotide selectivity in pyruvate kinase.

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

Data of fixed-target pink-beam serial synchrotron crystallography at the Pohang Light Source II.

Pink-beam serial synchrotron crystallography (SSX) is beneficial in terms of X-ray flux and overcoming partial reflection compared with SSX using a monochromatic beam. The fixed-target (FT) scanning method can minimize the physical damage on the crystal sample when delivering the crystals to the X-ray interaction point. Additionally, general researchers can easily access the experiment since no specialized sample transfer technology is needed. The fixed-target pink-beam SSX at the 1C beamline at the Pohang Light Source II (PLS-II) was previously demonstrated using a newly developed magnetic-based sample holder. The room-temperature structure of glucose isomerase and lysozyme were determined using FT pink-beam SSX. Meanwhile, the SSX dataset for glucose isomerase and lysozyme images containing the high X-ray background and multi-crystal hits. These data can be tentatively used to develop an indexing algorithm and practice processing the SX data. This study used detailed information on the diffraction data of fixed-target pink-beam SSX at PLS-II to access the raw data and process the information.

Structure Function Studies of Photosystem II Using X-Ray Free Electron Lasers.

The structure and mechanism of the water-oxidation chemistry that occurs in photosystem II have been subjects of great interest. The advent of X-ray free electron lasers allowed the determination of structures of the stable intermediate states and of steps in the transitions between these intermediate states, bringing a new perspective to this field. The room-temperature structures collected as the photosynthetic water oxidation reaction proceeds in real time have provided important novel insights into the structural changes and the mechanism of the water oxidation reaction. The time-resolved measurements have also given us a view of how this reaction-which involves multielectron, multiproton processes-is facilitated by the interaction of the ligands and the protein residues in the oxygen-evolving complex. These structures have also provided a picture of the dynamics occurring in the channels within photosystem II that are involved in the transport of the substrate water to the catalytic center and protons to the bulk.

Structural basis of homodimerization of the JNK scaffold protein JIP2 and its heterodimerization with JIP1.

The scaffold proteins JIP1 and JIP2 intervene in the c-Jun N-terminal kinase (JNK) pathway to mediate signaling specificity by coordinating the simultaneous assembly of multiple kinases. Using NMR, we demonstrate that JIP1 and JIP2 heterodimerize via their SH3 domains with the affinity of heterodimerization being comparable to homodimerization. We present the high-resolution crystal structure of the JIP2-SH3 homodimer and the JIP1-JIP2-SH3 heterodimeric complex. The JIP2-SH3 structure reveals how charge differences in residues at its dimer interface lead to formation of compensatory hydrogen bonds and salt bridges, distinguishing it from JIP1-SH3. In the JIP1-JIP2-SH3 complex, structural features of each homodimer are employed to stabilize the heterodimer. Building on these insights, we identify key residues crucial for stabilizing the dimer of both JIP1 and JIP2. Through targeted mutations in cellulo, we demonstrate a functional role for the dimerization of the JIP1 and JIP2 scaffold proteins in activation of the JNK signaling pathway.

Podophyllotoxin derivatives-tubulin complex reveals a potential binding site of tubulin polymerization inhibitors in α-tubulin adjacent to colchicine site.

The colchicine site of ß-tubulin has been proven to be essential binding sites of microtubule polymerization inhibitors. Recent studies implied that GTP pocket of α-tubulin adjacent to colchicine sites is a potential binding site for developing tubulin polymerization inhibitors. However, the structural basis for which type of structural fragments was more beneficial for enhancing the affinity of α-tubulin is still unclear. Here, podophyllotoxin derivatives-tubulin complex crystals indicated that heterocyclic with the highly electronegative and small steric hindrance was conducive to change configuration and enhance the affinity of the residues in GTP pocket of α-tubulin. Triazole with lone-pairs electrons and small steric hindrance exhibited the strongest affinity for enhancing affinity of podophyllotoxin derivatives by forming two hydrogen bonds with αT5 Ser178. Pyrimidine with the secondary strong affinity could bind Asn101 to make the αH7 configuration deflection, which reduces the stability of tubulin result in its depolymerization. Conversely, 4ß-quinoline-podophyllotoxin with the weakest affinity did not interact with α-tubulin. The molecular dynamics simulation and protein thermal shift results showed that 4ß-triazole-podophyllotoxin-tubulin was the most stable mainly due to two hydrogen bonds and the higher van der Waals force. This work provided a structural basis of the potential binding sites for extending the α/ß-tubulin dual-binding sites inhibitors design strategy.

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Šresolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

Binding of steroid substrates reveals the key to the productive transition of the cytochrome P450 OleP.

OleP is a bacterial cytochrome P450 involved in oleandomycin biosynthesis as it catalyzes regioselective epoxidation on macrolide intermediates. OleP has recently been reported to convert lithocholic acid (LCA) into murideoxycholic acid through a highly regioselective reaction and to unspecifically hydroxylate testosterone (TES). Since LCA and TES mainly differ by the substituent group at the C17, here we used X-ray crystallography, equilibrium binding assays, and molecular dynamics simulations to investigate the molecular basis of the diverse reactivity observed with the two steroids. We found that the differences in the structure of TES and LCA affect the capability of these molecules to directly form hydrogen bonds with N-terminal residues of OleP internal helix I. The establishment of these contacts, by promoting the bending of helix I, fosters an efficient trigger of the open-to-closed structural transition that occurs upon substrate binding to OleP and contributes to the selectivity of the subsequent monooxygenation reaction.

Major-groove sequence-specific RNA recognition by LoaP, a paralog of transcription elongation factor NusG.

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding ß-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.

Bridging the microscopic divide: a comprehensive overview of micro-crystallization and in vivo crystallography.

A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.

Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the ß-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.