High-resolution Structures Research Articles

Abstract 3883: Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia

Abstract The AAA ATPase p97, also known as valosin-containing protein (VCP), serves to regulate protein homeostasis by facilitating the translocation of ubiquitinated proteins from membranes or chromatin to the proteasome for degradation. Certain cancers, including multiple myeloma (MM) and acute myeloid leukemia (AML) are known for the intracellular overexpression of proteins, and are thus susceptible to the inhibition of p97 and resulting proteotoxic stress. A known p97 inhibitor, CB-5083 entered human phase 1 clinical trials in 2015, but trials were terminated due to side effects that were later traced to strong interaction of the compound with the phosphodiesterase PDE6. To address this problem, we devised a subtractive approach that leveraged high resolution structures of CB-5083 bound to PDE6 and p97. Using this information, we designed a p97 inhibitor that no longer displayed significant off-target binding but retained strong on-target binding. The optimization process started when we identified the 7-azaindole core from an in silico screen as a suitable hit pharmacophore and confirmed the binding mode of several variants of this scaffold in p97 by cryo-EM. Through a structure-guided medicinal chemistry program, we identified GND-135, a potent, on-target binder through the synthesis of less than 100 compounds. The design was supported with about 50 cryo-EM structures throughout the optimization process. Compared to CB-5083, our compound GND-135 displays greatly reduced PDE6 activity (< 1% inhibition at 10 μM versus 89% inhibition for CB-5083) and more potent p97 ATPase inhibition (3 nM IC50 versus 12 nM for CB-5083). We also evaluated the pharmacokinetic profile of GND-135 in mice; the half-life and clearance of 6.6 h and 15.9 mL/min/kg respectively were deemed suitable for daily administration. Based on the initial profiling, we carried out an efficacy study in a cell derived xenograft mouse model of AML using a U937 cell line with subcutaneous implantation. In this model, where GND-135 was administered IP (40 mg/kg QD) and CB-5083 was administered orally (40 mg/kg QD) the compounds showed statistically comparable efficacy, which was differentiated from untreated control. Thus, GND-135 is a novel p97 inhibitor lead compound with on-target selectivity powered by cryo-EM driven rational drug design. Citation Format: Jason Crawford, Ravi Munuganti, Charles Leung, Kriti Singh, Ellen Gates, Xing Zhu, Marcel Bally, Nancy Dos Santos, Maryam Sharifiaghdam, Zeynab Nosrati, Peter Axerio-Cilies, Alison Berezuk, Spencer Cholak, Sriram Subramaniam. Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3883.

Improving the Quality of Spontaneously Growing HviGH11 Crystals by Increasing the Viscosity Using Polyethylene Glycols

Proteins can form crystals spontaneously without crystallization experiments. These crystals can be used to determine three-dimensional structures. However, when X-ray diffraction is poor, crystal optimization is required to obtain a high-resolution crystal structure. Endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) spontaneously formed microcrystals after affinity purification and concentration; however, most HviGH11 microcrystals showed poor diffraction in the synchrotron X-ray and X-ray free-electron laser, so a complete three-dimensional structure could not be obtained. This study presents a method to improve the crystal quality of spontaneously grown HviGH11 microcrystals. The crystallization screening results revealed that temperature, pH, and salt were not crucial factors in increasing the solubility or preventing the spontaneous crystal growth of HviGH11. Conversely, the addition of polyethylene glycols (PEGs) as a precipitant facilitated the growth of larger HviGH11 crystals. The improved large HviGH11 crystal showed a diffraction of up to 1.95 Å when exposed to synchrotron X-rays, providing a complete three-dimensional structural dataset. Based on the nucleation rate equation, it was suggested that PEG increases the viscosity of the protein solution rather than promoting nucleation. This increase in viscosity reduced nucleation and facilitated the growth of larger HviGH11 crystals. These results provide valuable insights for future experiments aimed at increasing the size of spontaneously grown crystals.

Structural insight into Escherichia coli CsgA amyloid fibril assembly.

The discovery of functional amyloids in bacteria dates back several decades, and our understanding of the Escherichia coli curli biogenesis system has gradually expanded over time. However, due to its high aggregation propensity and intrinsically disordered nature, CsgA, the main structural component of curli fibrils, has eluded comprehensive structural characterization. Recent advancements in cryo-electron microscopy (cryo-EM) offer a promising tool to achieve high-resolution structural insights into E. coli CsgA fibrils. In this study, we outline an approach to addressing the colloidal instability challenges associated with CsgA, achieved through engineering and electrostatic repulsion. Then, we present the cryo-EM structure of CsgA fibrils at 3.62 Å resolution. This structure provides new insights into the cross-β structure of E. coli CsgA. Additionally, our study identifies two distinct spatial arrangements within several CsgA fibrils, a 2-CsgA-fibril pair and a 3-CsgA-fibril bundle, shedding light on the intricate hierarchy of the biofilm extracellular matrix and laying the foundation for precise manipulation of CsgA-derived biomaterials.IMPORTANCEThe visualization of the architecture of Escherichia coli CsgA amyloid fibril has been a longstanding research question, for which a high-resolution structure is still unavailable. CsgA serves as a major subunit of curli, the primary component of the extracellular matrix generated by bacteria. The support provided by this extracellular matrix enables bacterial biofilms to resist antibiotic treatment, significantly impacting human health. CsgA has been identified in members of Enterobacteriaceae, with pathogenic E. coli being the most well-known model system. Our novel insights into the structure of E. coli CsgA protofilaments form the basis for drug design targeting diseases associated with biofilms. Additionally, CsgA is widely researched in biomaterials due to its self-assembly characteristics. The resolved spatial arrangements of CsgA amyloids revealed in our study will further enhance the precision design of functional biomaterials. Therefore, our study uniquely contributes to the understanding of CsgA amyloids for both microbiology and material science.

Structural insights into the Clp protein degradation machinery.

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Denoising OCT videos based on temporal redundancy

The identification of eye diseases and their progression often relies on a clear visualization of the anatomy and on different metrics extracted from Optical Coherence Tomography (OCT) B-scans. However, speckle noise hinders the quality of rapid OCT imaging, hampering the extraction and reliability of biomarkers that require time series. By synchronizing the acquisition of OCT images with the timing of the cardiac pulse, we transform a low-quality OCT video into a clear version by phase-wrapping each frame to the heart pulsation and averaging frames that correspond to the same instant in the cardiac cycle. Here, we compare the performance of our one-cycle denoising strategy with a deep-learning architecture, Noise2Noise, as well as classical denoising methods such as BM3D and Non-Local Means (NLM). We systematically analyze different image quality descriptors as well as region-specific metrics to assess the denoising performance based on the anatomy of the eye. The one-cycle method achieves the highest denoising performance, increases image quality and preserves the high-resolution structures within the eye tissues. The proposed workflow can be readily implemented in a clinical setting.

Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius

Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).

3-D visual analysis and quantitative characterization on coal matrix based on Micro-CT

Coal matrix, the storage space for adsorbed gas, is highly related to gas recovery and gas outburst disasters. However, existing research predominantly relies on theoretical assumptions, lacking experimental validation. In this study, we employ X-ray computed tomography (CT) to obtain high-resolution (13.45–15.157 μm) internal structure images of various coal samples. The three-dimensional model of coal matrix elements is reconstructed using entropy threshold segmentation and watershed segmentation. A systematic quantification of the size, shape, and spatial arrangement of these matrix elements is then performed. The results reveal that coal matrix element equivalent diameters span 148.45–2800.35 μm, with volume-weighted average equivalent diameters ranging from 1320.82 to 1720.87 μm. Additionally, we explore particle size distribution (PSD) models to characterize size-quantity relationships and identify the Jaky model based on the logarithmic-exponential equation as the optimal PSD model. Observation of the matrix element models shows that large matrix elements typically exhibit flattened morphologies with rough surfaces, while smaller ones tend to be blocky with smooth surfaces. The dimensionless shape factor results suggest that, when scale effects are not considered, the shape of larger matrix elements is more conducive to matrix-fracture gas mass transfer. Based on this, a functional relationship between the matrix shape factor and size, considering changes in matrix morphology, is established. Moreover, we algorithmically determine that the average number of adjacent matrix elements per individual matrix element falls between 13 and 15. For matrix clusters with large volume fractions, the size ratio of the central matrix element to adjacent matrix elements primarily falls within the range of 0.4–0.6, approximately following a Weibull distribution. According to this feature, a method for building a matrix cluster model used for simulations is proposed.

Targeting imidazole-glycerol phosphate dehydratase in plants: novel approach for structural and functional studies, and inhibitor blueprinting.

The histidine biosynthetic pathway (HBP) is targeted for herbicide design with preliminary success only regarding imidazole-glycerol phosphate dehydratase (IGPD, EC 4.2.1.19), or HISN5, as referred to in plants. HISN5 catalyzes the sixth step of the HBP, in which imidazole-glycerol phosphate (IGP) is dehydrated to imidazole-acetol phosphate. In this work, we present high-resolution cryoEM and crystal structures of Medicago truncatula HISN5 (MtHISN5) in complexes with an inactive IGP diastereoisomer and with various other ligands. MtHISN5 can serve as a new model for plant HISN5 structural studies, as it enables resolving protein-ligand interactions at high (2.2 Å) resolution using cryoEM. We identified ligand-binding hotspots and characterized the features of plant HISN5 enzymes in the context of the HISN5-targeted inhibitor design. Virtual screening performed against millions of small molecules not only revealed candidate molecules but also identified linkers for fragments that were experimentally confirmed to bind. Based on experimental and computational approaches, this study provides guidelines for designing symmetric HISN5 inhibitors that can reach two neighboring active sites. Finally, we conducted analyses of sequence similarity networks revealing that plant HISN5 enzymes derive from cyanobacteria. We also adopted a new approach to measure MtHISN5 enzymatic activity using isothermal titration calorimetry and enzymatically synthesized IGP.

Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.

High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.

Linking the Autotaxin-LPA Axis to Medicinal Cannabis and the Endocannabinoid System.

Over the past few decades, many current uses for cannabinoids have been described, ranging from controlling epilepsy to neuropathic pain and anxiety treatment. Medicines containing cannabinoids have been approved by both the FDA and the EMA for the control of specific diseases for which there are few alternatives. However, the molecular-level mechanism of action of cannabinoids is still poorly understood. Recently, cannabinoids have been shown to interact with autotaxin (ATX), a secreted lysophospholipase D enzyme responsible for catalyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a pleiotropic growth factor that interacts with LPA receptors. In addition, a high-resolution structure of ATX in complex with THC has recently been published, accompanied by biochemical studies investigating this interaction. Due to their LPA-like structure, endocannabinoids have been shown to interact with ATX in a less potent manner. This finding opens new areas of research regarding cannabinoids and endocannabinoids, as it could establish the effect of these compounds at the molecular level, particularly in relation to inflammation, which cannot be explained by the interaction with CB1 and CB2 receptors alone. Further research is needed to elucidate the mechanism behind the interaction between cannabinoids and endocannabinoids in humans and to fully explore the therapeutic potential of such approaches.

Structure of the human 20S U5 snRNP.

The 20S U5 small nuclear ribonucleoprotein particle (snRNP) is a 17-subunit RNA-protein complex and a precursor of the U4/U6.U5 tri-snRNP, the major building block of the precatalytic spliceosome. CD2BP2 is a hallmark protein of the 20S U5 snRNP, absent from the mature tri-snRNP. Here we report a high-resolution cryogenic electron microscopy structure of the 20S U5 snRNP, shedding light on the mutually exclusive interfaces utilized during tri-snRNP assembly and the role of the CD2BP2 in facilitating this process.

Fault-scale crustal structure across the Dunhua-Mishan fault (Tanlu northern segment) constrained from teleseismic P-wave receiver functions

The Dunhua-Mishan fault, located in the northern segment of the Tanlu fault zone, experienced multiple tectonic processes associated with the effects of the Pacific Plate subduction and the Indo-Asia collision. The high-resolution fault-scale structure is critical for understanding the fault evolution and potential fault damage. However, the well-defined deep structure of the Dunhua-Mishan fault is still unclear due to the lack of the dense seismic array. In this study, we construct a high-resolution P-wave receiver function imaging based on linear dense seismic array across the fault. Our results reveal the strong Moho depth variation across the Dunhua-Mishan fault zone. The slightly higher Vp/Vs ratio values within the fault zone indicate the presence of a small amount of mafic crust composition. Interestingly, the significant double positive Ps converted phases are observed within the fault zone, which may represent double Moho discontinuities. The double Moho structure may be related to multiple significant tectonic activities in the Tanlu northern segment. These newly observed structures provide new seismic constraints on the formation and evolution of the Tanlu fault zone and probably reflect that the lithospheric structure of the Dunhua-Mishan fault has been modified by a series of tectonic processes.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Structure of the human outer kinetochore KMN network complex

Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.

Cryo-EM structure of wheat ribosome reveals unique features of the plant ribosomes

Plants being sessile organisms exhibit unique features in ribosomes, which might aid in rapid gene expression and regulation in response to varying environmental conditions. Here, we present high-resolution structures of the 60S and 80S ribosomes from wheat, a monocot staple crop plant (Triticum aestivum). While plant ribosomes have unique plant-specific rRNA modification (Cm1847) in the peptide exit tunnel (PET), the zinc-finger motif in eL34 is absent, and uL4 is extended, making an exclusive interaction network. We note differences in the eL15-helix 11 (25S) interaction, eL6-ES7 assembly, and certain rRNA chemical modifications between monocot and dicot ribosomes. In eukaryotes, we observe highly conserved rRNA modification (Gm75) in 5.8S rRNA and a flipped base (G1506) in PET. These features are likely involved in sensing or stabilizing nascent chain. Finally, we discuss the importance of the universal conservation of three consecutive rRNA modifications in all ribosomes for their interaction with A-site aminoacyl-tRNA.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Structural insight into an Arl1–ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin

Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1–Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1–Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1–Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1–Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.