A Sialic Acid Binding Site in a Human Picornavirus

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.     

Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.     

Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site

In maize chloroplasts, the ratio of HCO₃⁻ (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO₃⁻ to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO₃⁻ and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO₃⁻ binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO₃⁻ with a dissociation constant near 80 micromolar. The location of the HCO₃⁻ binding site, and the low-affinity atrazine binding site, is not known.     

The Yeast E4 Ubiquitin Ligase Ufd2 Interacts with the Ubiquitin-like Domains of Rad23 and Dsk2 via a Novel and Distinct Ubiquitin-like Binding Domain

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.     

Altered Microviscosity at Brain Membrane Surface Induces Distinct and Reversible Inhibition of Opioid Receptor Binding

In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis-delta 6) acid, oleic (18:1, cis-delta 9) acid, and vaccenic (18:1, cis-delta 11) acid or their corresponding methyl esters at 0.5 mumol/mg of membrane protein caused a similar 7-10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5-7% by the fatty acids but only 1% by their methyl esters. Concomitantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated mu-, delta-, and kappa-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order delta greater than mu much greater than kappa, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5-3 mumol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited mu-, delta-, and kappa-opioid binding toward control values. In particular, the Bmax of [3H]-DPDPE binding completely recovered after being undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.     

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 10⁶, 10⁷ or, 10⁸ CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 10⁷ or 10⁸ CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16.h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Protein binding is an important component of pharmacokinetic/pharmacodynamic (PK/PD) research. In vitro measurement of protein drug binding is an essential component of the research and development of novel drugs. However, in vitro studies often poorly mirror the in vivo condition.^9,42 Pharmacokinetic studies early in drug development provide a means to assess the time course of drug effects in the body and drug distribution and availability.⁴² From a PK/PD modeling perspective, protein binding is an important factor in the kinetics and dynamics of drug availability in vivo.^21,35,36,40 These complex relationships are used to project efficacious doses in humans and take into consideration differences in plasma protein binding between preclinical species and humans.^8,44A variety of acute phase proteins (APP) exist across all species and increase in response to inflammatory, infectious and traumatic events.^{5,9,12,13,19,21,22,29,45,53} APPs are potential biomarkers for detection and monitoring of various disease states including cancer.^{2,18,24,34,39,40,47,50,52} Because of this, enhanced understanding of drug binding characteristics to APPs early in the development phase will promote the design of more efficacious therapeutics. Alpha-1 acid glycoprotein (AGP), a ubiquitous major APP that is present in rats,^9,46 has significant drug binding properties and binds to many basic and neutral compounds. Normal AGP levels in plasma of naïve rats range from 0.1 to 0.32 mg/mL.⁴⁴ The importance of AGP as related to drug discovery and development will be bolstered by greater understanding of the sources of AGP stimulation in established animal models. For example, AGP modulates the immune response in a rodent shock model in which it is thought to maintain normal capillary permeability to ensure perfusion of vital organs.^30,33 In addition, elevated AGP levels are present in animal models of infection and inflammation.^{11,20,27,32,41,48}In surgically modified animals, AGP levels may be elevated after surgical manipulation, which unavoidably induces local transient inflammatory responses.^8,25,51 In addition, infections may develop postoperatively leading to increased AGP levels. Chronic catheterization has been linked to increased incidence of infection.^3,8,37 Surgically modified animals should not be placed on study if aseptic technique was not adhered to during surgical preparation and instrumentation.^6,37 Contamination may occur within or at the external portion of a catheter, usually resulting in more obvious signs of infection. Routine PK studies in rats involve implantation of vascular catheters through which drugs are administered and blood samples are taken over time. Catheterized animals are typically perceived as being healthy and thus are enrolled in and remain on study unless they develop obvious clinical signs of infection or illness. However, an occult infection may be present even with a patent catheter. As such, understanding the direct effect of subclinical infection in modulating AGP levels and drug binding is critical, as AGP levels may affect drug levels in study animals with persistent subclinical infection. In this event, the PK data generated may be altered due to selective binding to AGP, thus confounding data interpretation.A possible application of AGP is its potential utility as a biomarker for evaluating health status animals in drug development. The use of AGP as a select biomarker for monitoring and identifying sick animals and/or predicting the potential impact of subclinical infection on drug PK/PD is highly desirable. A screening tool such as this could help to optimize animal selection by reliably identifying healthy animals. Improved intra-study health monitoring would promote confidence in PK/PD data and its predictive value.The focus of this research was to develop a sensitive, reliable and reproducible model of subclinical infection in the rat using the ubiquitous skin contaminant, S. aureus. We selected AGP as a biomarker that would promote health status screening and enhance PK/PD characterization of AGP binding drugs (that is basic and neutral) in the presence or absence of subclinical infection. The model was validated by evaluating the impact of increased AGP levels on propranolol, a drug known to have high binding affinity to AGP.^{4,7,10,26,28,31,49} Ultimately, establishing this model will provide heightened visibility of the protein binding characteristics of drugs and yield more informed data interpretation.     

Binding of Lipoic Acid Induces Conformational Change and Appearance of a New Binding Site in Methylglyoxal Modified Serum Albumin

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1

An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposi's sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1DeltaTMFc). K8.1DeltaTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1DeltaTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.     

果蝇TRP离子通道C端一个新钙调蛋白结合位点的鉴定和分析

瞬时受体电位(TRP)通道是一类钙离子透过性的阳离子通道蛋白家族,参与了视觉、味觉、温度感受等重要的生物学过程。之前的研究表明,钙离子既能够正反馈也能够负反馈地调节瞬时受体电位通道的活性,而这种调节可能是通过钙调蛋白（calmodulin,CaM）与TRP通道的相互作用来进行的。为了阐明这一调控机制,我们首先需要对钙调蛋白与瞬时受体电位通道之间的相互作用进行详细的生化研究。在此项研究中,通过大肠杆菌表达系统,表达和纯化了果蝇瞬时受体电位通道羧基末端不同长短的蛋白片段,并发现了一个新的钙调蛋白结合位点。通过快速蛋白液相色谱、静态光散射以及等温量热滴定技术,鉴定了这一钙调蛋白结合位点与果蝇瞬时受体电位通道之间的相互作用,发现它们在钙离子依赖的条件下,可以形成亲和力非常强的稳定的蛋白复合物（解离常数在01～1微摩尔范围）。此外,通过合成多肽的方法,鉴定了果蝇瞬时受体电位通道913～939片段为该钙调蛋白结合位点的核心区域。最后,通过突变实验,进一步明确了果蝇瞬时受体电位通道922位的酪氨酸以及923位的缬氨酸为其钙调蛋白结合位点的关键氨基酸。总而言之,本研究发现和鉴定了果蝇瞬时受体电位通道上一个新的钙依赖的钙调蛋白结合位点,这一发现将为研究瞬时受体电位通道的体内功能提供生化基础,为阐明钙离子通过钙调蛋白调节瞬时受体电位通道的分子机制做出贡献。     

Crystal Structure of Transglutaminase 2 with GTP Complex and Amino Acid Sequence Evidence of Evolution of GTP Binding Site

Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.     

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.     

Somatosensory Abnormalities for Painful and Innocuous Stimuli at the Back and at a Site Distinct from the Region of Pain in Chronic Back Pain Patients

Chronic low back pain (CLBP) was shown to be associated with pathophysiological changes at several levels of the sensorimotor system. Changes in sensory thresholds have been reported but complete profiles of Quantitative Sensory Testing (QST) were only rarely obtained in CLBP patients. The aim of the present study was to investigate comprehensive QST profiles in CLBP at the painful site (back) and at a site distinct from their painful region (hand) and to compare these data with similar data in healthy controls. We found increased detection thresholds in CLBP patients compared to healthy controls for all innocuous stimuli at the back and extraterritorial to the painful region at the hand. Additionally, CLBP patients showed decreased pain thresholds at both sites. Importantly, there was no interaction between the investigated site and group, i.e. thresholds were changed both at the affected body site and for the site distinct from the painful region (hand). Our results demonstrate severe, widespread changes in somatosensory sensitivity in CLBP patients. These widespread changes point to alterations at higher levels of the neuraxis or/and to a vulnerability to nociceptive plasticity in CLBP patients.