Yersinia infection tools-characterization of structure and function of adhesins

Peer reviewe

Structure of the UspA1 protein fragment from Moraxella catarrhalis responsible for C3d binding

The gram-negative bacterium Moraxella catarrhalis infects humans exclusively, causing various respiratory tract diseases, including acute otitis media in children, septicaemia or meningitis in adults, and pneumonia in the elderly. To do so, M. catarrhalis expresses virulence factors facilitating its entry and survival in the host. Among them are the ubiquitous surface proteins (Usps): A1, A2, and A2H, which all belong to the trimeric autotransporter adhesin family. They bind extracellular matrix molecules and inhibit the classical and alternative pathways of the complement cascade by recruiting complement regulators C3d and C4b binding protein. Here, we report the 2.5 Å resolution X-ray structure of UspA1299-452, which previous work had suggested contained the canonical C3d binding site found in both UspA1 and UspA2. We show that this fragment of the passenger domain contains part of the long neck domain (residues 299 – 336) and a fragment of the stalk (residues 337 – 452). The coiled-coil stalk is left-handed, with 7 polar residues from each chain facing the core and coordinating chloride ions or water molecules. Despite the previous reports of tight binding in serum-based assays, we were not able to demonstrate binding between C3d and UspA1299-452 using ELISA or biolayer interferometry, and the two proteins run separately on size-exclusion chromatography. Microscale thermophoresis suggested that the dissociation constant was 140.5 ± 8.4 μM. We therefore suggest that full-length proteins or other additional factors are important in UspA1-C3d interactions. Other molecules on the bacterial surface or present in serum may enhance binding of those two molecules.Peer reviewe

The crystal structure of PD1, a Haemophilus surface fibril domain

The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis.Peer reviewe

Total Lean Body Mass and Lower Body Lean Mass Correlation with Vertical Jump in Untrained Women Basketball Players after 8 Weeks of Resistance Training

Basketball is a fast, explosive sport where a high vertical jump (VJ) is extremely beneficial. Research has shown that greater amounts of lean mass have been associated with higher force output, and therefore higher VJ. PURPOSE: The purpose of this study is to examine the correlation of overall total lean body mass percentage (TBLM%) and lower body lean mass percentage (LBLM%) and Body fat percentage (BF%) with VJ in previously untrained collegiate D-III women basketball players. METHODS: Fourteen females (20±1.3 years, 170.68±8.76 cm, T1 BF% 28.68±5.38, T2 27.11±5.12) basketball team participated in this study. Athletes were tested on two different occasions (T1 and T2). Dual-X-Ray Absorptiometry (DXA) body composition scan and standard VJ assessment was performed using a Vertec to determine maximum jump height prior to (T1) and following (T2) 8 weeks of an undulating periodization resistance training program 5 days/week). DXA were further analyzed to determine total body lean mass % (TBLM%) and lower-body lean mass % (LBLM%). Data was analyzed using SPSS using paired samples T-test to detect differences (

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the “common microbial binding site” on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.Peer reviewe

Crystal structure of human cystatin C stabilized against amyloid formation

Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain-like (C1) and legumain-related (C13) cysteine proteases. In pathophysiological processes, the nature of which is not understood, HCC is codeposited in the amyloid plaques of Alzheimer's disease or Down's syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life. In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain-swapped dimers, created through a conformational change of a beta-hairpin loop, L1, from the papain-binding epitope. We have created monomer-stabilized human cystatin C, with an engineered disulfide bond (L47C)-(G69C) between the structural elements that become separated upon domain swapping. The mutant has drastically reduced dimerization and fibril formation properties, but its inhibition of papain is unaltered. The structure confirms the success of the protein engineering experiment to abolish 3D domain swapping and, in consequence, amyloid fibril formation. It illustrates for the first time the fold of monomeric cystatin C and allows verification of earlier predictions based on the domain-swapped forms and on the structure of chicken cystatin. Importantly, the structure defines the so-far unknown conformation of loop L1, which is essential for the inhibition of papain-like cysteine proteases

Structural studies of human muscle FBPase

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg$^{2+}$, Mn$^{2+}$, or Zn$^{2+}$, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of –85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS)

Observation of Jump Height, Peak Propulsive Force, Braking Force, and Loading Force in Acrobatics and Tumbling Athletes During a Completive Season: Pilot

Acrobatics and tumbling is a women’s sport comprised of 3 different positions: base, top, and tumbler. Each position endures substantial impact on the musculotendinous unit, however, tumblers experience the greatest lower extremity eccentric impact leading to a high injury rate. Consistent athlete monitoring may lead to injury prevention and better insights into training. PURPOSE: The purpose of this study is to pilot observational differences in jump height (JH), peak propulsive force (PPF), peak braking force (PBF), and peak landing force (PLF) during the counter movement jump (CMJ) in acrobatics and tumbling between tumbler and non-tumbler athletes during a competitive season. METHODS: Thirty-three female acrobatics and tumbling athletes volunteered for this study and performed jump testing 3 days per week during their competition season. Data was filtered to include only subjects (n = 15; 19.6 ± 1.0 yrs, 160.7 ± 7.156 cm; 63.5 ± 17.9 kg) ten tumblers (160.12 ± 7.02 cm; 57.5 ± 19.1 kg) and five non-tumblers (162.2 ± 3.27 cm; 71.8 ± 10.0 kg) that had consistent compliance during the six-week period. Participants performed three CMJs using Hawkin Dynamics force plates and software with each CMJ separated by a ten second rest. Data was analyzed using SPSS using a 2X6 (position X time) ANOVA (pRESULTS: No significant main effects for time were found (p=0.95) and there was no significant interaction between time and position (p=0.97). There was a significant main effect for position (p=0.006). Follow up analysis observed significant differences in JH, PPF and PBF (pCONCLUSION: This was a pilot study to observe changes eccentric and concentric loading throughout the competitive season of a DIII acrobatics and tumbling team. When collapsed across time, tumblers experienced greater decline in JH, PPF and PBF throughout the season