Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation

Structural mapping of oligomeric intermediates in an amyloid assembly pathway

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human β2-microglobulin (β2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-?-D-glucoside (?-OG), n-dodecyl-?-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR

¹H, ¹?N, and ¹³C backbone chemical shift assignment of titin domains A59-A60 and A60 alone

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is > 1 ?m long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or 'super-repeats'. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619-628, 2012). Here we report backbone (15)N, (13)C and (1)H chemical shift and (13)C? assignments for the A59-A60 domain tandem from the titin A59-A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59-A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59-A60

Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly

In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of ?-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation

The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A.

Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate ?-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor

Distinguishing closely related amyloid precursors using an RNA aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ?2-microglobulin (h?2m) can co-polymerize with its N-terminally truncated variant (?N6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using h?2m and its amyloidogenic counterpart, ??6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds h?2m with an EC50 of ?200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-h?2m complex, revealing that the aptamer binds to the face of h?2m containing the A, B, E, and D ?-strands. In contrast, binding of B6 to ?N6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of h?2m and ?N6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity

Mechanisms of amyloid formation revealed by solution NMR

Amyloid fibrils are proteinaceous elongated aggregates involved in more than fifty human diseases. Recent advances in electron microscopy and solid state NMR have allowed the characterization of fibril structures to different extents of refinement. However, structural details about the mechanism of fibril formation remain relatively poorly defined. This is mainly due to the complex, heterogeneous and transient nature of the species responsible for assembly; properties that make them difficult to detect and characterize in structural detail using biophysical techniques. The ability of solution NMR spectroscopy to investigate exchange between multiple protein states, to characterize transient and low-population species, and to study high molecular weight assemblies, render NMR an invaluable technique for studies of amyloid assembly. In this article we review state-of-the-art solution NMR methods for investigations of: (a) protein dynamics that lead to the formation of aggregation-prone species; (b) amyloidogenic intrinsically disordered proteins; and (c) protein–protein interactions on pathway to fibril formation. Together, these topics highlight the power and potential of NMR to provide atomic level information about the molecular mechanisms of one of the most fascinating problems in structural biology