RESUMEN
Lipase is a versatile enzyme found in microorganisms, animals and plants. It has applications in a wide variety of fields ranging from the food industry to the pharmaceutical. For these applications, mainly microbial lipases are exploited in great detail. On the other hand lipases from the plant source have been characterized to a much lesser extent. Although many plant lipase sequences have been reported in UniProtKB, till date there is no report on the crystal structure of any plant lipase. In view of very limited availability of structural information on plant lipases, in this study, we modeled the three-dimensional structure of seven plant lipases and studied the conformational changes under four different solvents at two different temperatures. Most lipases have a lid domain and its movement is implicated in the interfacial activation of lipases. Among the 56 conditions tested in this study, some lipases at certain condition exhibit the lid domain movement thus implying the functional importance. Laborious purification and minimal yield are the likely reasons for poor characterization of plant lipases. In this scenario, the results of computational studies on plant lipases under different environmental conditions will provide useful data for subsequent in vitro functional studies.
Asunto(s)
Lipasa/química , Simulación de Dinámica Molecular , Proteínas de Plantas/química , Conformación Proteica , Solventes/química , Temperatura , Secuencia de Aminoácidos , Fenómenos Químicos , Dominios Proteicos , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).
Asunto(s)
Adhesinas Bacterianas , Adhesión Bacteriana/efectos de los fármacos , Nanopartículas del Metal , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/biosíntesis , Adhesinas Bacterianas/efectos de los fármacos , Adhesinas Bacterianas/aislamiento & purificación , Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Fibrinógeno/efectos de los fármacos , Fibrinógeno/metabolismo , Humanos , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Unión Proteica , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
PfbA (Plasmin(ogen) and Fibronectin Binding protein A) is an adhesin present on the surface of Streptococcus pneumoniae. Initial studies characterized PfbA as plasmin(ogen) and fibronectin binding protein and later it was found that it binds with many other proteins of the extracellular matrix such as fibrinogen, collagen and laminin. It also binds to blood protein human serum albumin (HSA). Interestingly, PfbA exhibits no binding with serum albumins of bovine (BSA), rabbit (RSA) and porcine (PSA) which are sequentially and structurally homologous to HSA. This suggests that PfbA is likely involved in host specificity. Therefore, to get more insights into this aspect, a detailed analysis, which includes the interaction of rPfbA with HSA/BSA/RSA/PSA at different pHs by bio-layer interferometry, comparison of sequences and surface electrostatic potential of HSA/BSA/RSA/PSA, lysine modification of HSA by succinylation and subsequent interaction analysis of succinylated HSA with rPfbA and the secondary structural content estimation by FT-IR spectroscopy was carried out. Since large protrusions are another important geometric feature of protein surfaces, the property was also analyzed for HSA/BSA/RSA/PSA. The results of the above studies clearly suggest that the rPfbA exhibits host specificity by selectively binding only to HSA and not with its homologous BSA/RSA/PSA. Since the three dimensional structures of these albumins are highly similar, it is likely that rPfbA utilizes the differences in the surface electrostatic charge in combination with surface protrusions of HSA/BSA/RSA/PSA for the selective molecular recognition process and this feature may be important in the pathogenesis of pneumococcal infection.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Albúmina Sérica/metabolismo , Streptococcus pneumoniae/metabolismo , Animales , Bovinos , Especificidad del Huésped , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Conejos , Electricidad Estática , PorcinosRESUMEN
GAPDH being a key enzyme in the glycolytic pathway is one of the surface adhesins of many Gram-positive bacteria including Streptococcus agalactiae. This anchorless adhesin is known to bind to host plasminogen (PLG) and fibrinogen (Fg), which enhances the virulence and modulates the host immune system. The crystal structure of the recombinant GAPDH from S. agalactiae (SagGAPDH) was determined at 2.6â¯Å resolution by molecular replacement. The structure was found to be highly conserved with a typical NAD binding domain and a catalytic domain. In this paper, using biolayer interferometry studies, we report that the multifunctional SagGAPDH enzyme binds to a variety of host molecules such as PLG, Fg, laminin, transferrin and mucin with a KD value of 4.4â¯×â¯10-7â¯M, 9.8â¯×â¯10-7â¯M, 1â¯×â¯10-5â¯M, 9.7â¯×â¯10-12â¯M and 1.4â¯×â¯10-7â¯M respectively. The ligand affinity blots reveal that SagGAPDH binds specifically to α and ß subunits of Fg and the competitive binding ELISA assay reveals that the Fg and PLG binding sites on GAPDH does not overlap each other. The PLG binding motif of GAPDH varies with organisms, however positively charged residues in the hydrophobic surroundings is essential for PLG binding. The lysine analogue competitive binding assay and lysine succinylation experiments deciphered the role of SagGAPDH lysines in PLG binding. On structural comparison with S. pneumoniae GAPDH, K171 of SagGAPDH is being predicted to be involved in PLG binding. Further SagGAPDH exhibited enzymatic activity in the presence of Fg, PLG and transferrin. This suggests that these host molecules does not mask the active site and bind at some other region of GAPDH.