ABSTRACT
Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase that plays a crucial role in cell migration, chemotaxis, oxidative burst and myocardial contractility. It is activated downstream of G protein-coupled receptors (GPCRs) and small GTPases of Ras superfamily. PI3Kγ is a heterodimer composed of a catalytic and a regulatory subunit that is expressed mostly in hematopoietic cells and in the heart. Although it has attracted a lot of attention because of its link with tumor inflammation and heart diseases, its regulation is still not fully understood. This can be attributed to the absence of high-resolution structural details of the PI3Kγ heterodimer. Here we describe the design and purification of PI3Kγ constructs where flexible loops in the regulatory subunit have been removed based on structural information obtained by hydrogen/deuterium exchange - mass spectrometry (HDX-MS). The soluble constructs retain both basal activity and sensitivity to GPCR stimulation, and are thus an optimal tool to further explore their regulation using a structure-based approach.
Subject(s)
Class Ib Phosphatidylinositol 3-Kinase/genetics , Plasmids/metabolism , Protein Engineering/methods , Receptors, G-Protein-Coupled/genetics , Amino Acid Sequence , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Cloning, Molecular , Deuterium Exchange Measurement , Gene Expression , Humans , Mass Spectrometry , Plasmids/chemistry , Protein Multimerization , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , Spodoptera , SwineABSTRACT
Aspergillus niger alpha-amylase catalyses the hydrolysis of alpha-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P2(1)2(1)2(1) with one molecule per asymmetric unit and one belongs to the monoclinic space group P2(1) with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger alpha-amylase crystallized in the monoclinic space group P2(1) with two molecules per asymmetric unit in complex with maltose at 1.8 angstroms resolution is reported. Furthermore, a novel 1.6 angstroms resolution orthorhombic crystal form (space group P2(1)2(1)2) of the native enzyme is presented. Four maltose molecules are observed in the maltose-alpha-amylase complex. Three of these occupy active-site subsites -2 and -1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products.
