Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Mimicking Protein Kinase C Phosphorylation Inhibits Arc/Arg3.1 Palmitoylation and Its Interaction with Nucleic Acids.

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-ß2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-ß2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-ß2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-ß2 W373 corresponds to close paralog Karyopherin-ß2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-ß2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum.

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.

SViMULATE: a computer program facilitating interactive, multi-mode simulation of analytical ultracentrifugation data.

The ability to simulate sedimentation velocity (SV) analytical ultracentrifugation (AUC) experiments has proved to be a valuable tool for research planning, hypothesis testing, and pedagogy. Several options for SV data simulation exist, but they often lack interactivity and require up-front calculations on the part of the user. This work introduces SViMULATE, a program designed to make AUC experimental simulation quick, straightforward, and interactive. SViMULATE takes user-provided parameters and outputs simulated AUC data in a format suitable for subsequent analyses, if desired. The user is not burdened by the necessity to calculate hydrodynamic parameters for simulated macromolecules, as the program can compute these properties on the fly. It also frees the user of decisions regarding simulation stop time. SViMULATE features a graphical view of the species that are under simulation, and there is no limit on their number. Additionally, the program emulates data from different experimental modalities and data-acquisition systems, including the realistic simulation of noise for the absorbance optical system. The executable is available for immediate download.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 is linked to neurodevelopmental disorders.

The normally nuclear HNRNPH2 is mutated in HNRNPH2 -related X-linked neurodevelopmental disorder causing the protein to accumulate in the cytoplasm. Interactions of HNRNPH2 with its importin Karyopherin-ß2 (Transportin-1) had not been studied. We present a structure that shows Karyopherin-ß2 binding HNRNPH2 residues 204-215, a proline-tyrosine nuclear localization signal or PY-NLS that contains a typical R-X 2-4 -P-Y motif, 206 RPGPY 210 , followed a new Karyopherin-ß2 binding epitope at 211 DRP 213 that make many interactions with Karyopherin-ß2 W373. Mutations at each of these sites decrease Karyopherin-ß2 binding affinities by 70-100 fold, explaining aberrant accumulation in cells and emphasizing the role of nuclear import defects in the disease. Sequence/structure analysis suggests that the new epitope C-terminal of the PY-motif, which binds Karyopherin-ß2 W373, is rare and thus far limited to close paralogs HNRNPH2, HNRNPH1 and HNRNPF. Karyopherin-ß2 W373, a HNRNPH2-binding hotspot, corresponds to W370 of close paralog Transportin-2, a site of pathological variants in patients with neurodevelopmental abnormalities, suggesting that Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities. Summary: HNRNPH2 variants in HNRNPH2 -related X-linked neurodevelopmental disorder aberrantly accumulate in the cytoplasm. A structure of Karyopherin-ß2•HNRNPH2 explains nuclear import defects of the variants, reveals a new NLS epitope that suggests mechanistic changes in pathological variants of Karyopherin-ß2 paralog Transportin-2.

Self-assembling Peptides with Internal Ionizable Unnatural Amino Acids: A General Approach to pH-responsive Peptide Materials.

Self-assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH-responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH-responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain. Through a combined experimental and computational investigation, we demonstrate that these residues can be accommodated and stabilized within the internal hydrophobic compartment of the peptide assembly. The hydrophobic microenvironment shifts their pKa significantly from a basic pH typically found for free amines to a more biologically relevant pH in the weakly acidic range. The pH-induced ionization and ionization-dependent self-assembly and disassembly are thoroughly investigated and correlated with the biological activity of the assembly. This new approach has unique advantages in tuning the pH-responsiveness of self-assembled peptides across a large pH range in a complex biological environment. We anticipate the ionizable amino acids developed here can be widely applicable to the synthesis and self-assembly of many amphiphilic peptides with endowed pH-responsive properties to enhance their biological activities toward applications ranging from targeted therapeutic delivery to proton transport.

The feasibility of determining kinetic constants from isothermal titration calorimetry data.

Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on exploiting the power/time trace (the "thermogram") resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter. Thus, several critical factors must be taken into account: the injection protocol, the kinetics of the interaction, accurate discovery of the instrumental response to heat signals, and the addition of unrelated signals. All of these aspects of extracting kinetic constants from thermograms have been considered and addressed in the current work. To validate the resultant methods, we performed several ITC experiments, titrating small-molecule inhibitors into solutions of bovine carbonic anhydrase II or titrating lysozyme into solutions of anti-lysozyme nanobodies. We found that our methods could arrive at kinetic constants that were close to the known values for these interactions taken from other methods. Finally, the effort to improve ITC kinetic characterizations uncovered a set of best practices for both the calorimetric experiment and the subsequent analyses (termed "kinetically optimized ITC" or "KO-ITC") that is detailed in this work.

A Protein Semisynthesis-Based Strategy to Investigate the Functional Impact of Linker Histone Serine ADP-Ribosylation.

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study poly(ADP-ribose) polymerase (PARP) biology. Here, we establish a generalizable strategy to prepare ADP-ribosylated peptides that are compatible with N-terminal, C-terminal, and sequential protein ligation reactions. Two unique protein-assembly routes are employed to generate full-length linker histone constructs that are homogeneously ADP-ribosylated at known DNA damage-dependent modification sites. We found that serine mono-ADP-ribosylation is sufficient to alleviate linker histone-dependent chromatin compaction and that this effect is amplified by ADP-ribose chain elongation. Our work will greatly expand the scope of ADP-ribose-modified proteins that can be constructed via semisynthesis, which is rapidly emerging as a robust approach to elucidate the direct effects that site-specific serine mono- and poly-ADP-ribosylation have on protein function.

Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance.

Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN-transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post-translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane. As such, Ftp inhibitors potentially represent a new class of antimicrobials. Previously, we showed that AMP is both a product of the Ftp_tp-catalyzed reaction and an inhibitor of the enzyme. As a preliminary step in exploiting this property to develop a novel Ftp_Tp inhibitor, we have used structural and solution studies to examine the inhibitory and enzyme-binding properties of several adenine-based nucleosides, with particular focus on the 2-position of the purine ring. Implications for future drug design are discussed.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Measuring the KD of Protein-Ligand Interactions Using Microscale Thermophoresis.

Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein-ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.

Osmosensing by WNK Kinases.

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Biophysical and Biochemical Characterization of TP0037, a d-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum.

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism's robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein's propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum's d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidumIMPORTANCE Because T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway (TP0037; d-lactate dehydrogenase) requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.

Using modern approaches to sedimentation velocity to detect conformational changes in proteins.

It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called "difference SV" was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.

Tissue-Specific Regulation of the Wnt/ß-Catenin Pathway by PAGE4 Inhibition of Tankyrase.

Spatiotemporal control of Wnt/ß-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/ß-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the ß-catenin destruction complex. Although Wnt/ß-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/ß-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/ß-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/ß-catenin pathway.