Thermodynamics of Binding by Calmodulin Correlates with Target Peptide α-Helical Propensity

In this work, we have examined contributions to the thermodynamics of calmodulin (CaM) binding from the intrinsic propensity for target peptides to adopt an α-helical conformation. CaM target sequences are thought to commonly reside in disordered regions within proteins. Using the ability of TFE to induce α-helical structure as a proxy, the six peptides studied range from having almost no propensity to adopt α-helical structure through to a very high propensity. This despite all six peptides having similar CaM-binding affinities. Our data indicate there is some correlation between the deduced propensities and the thermodynamics of CaM binding. This finding implies that molecular recognition features, such as CaM target sequences, may possess a broad range of propensities to adopt local structure. Given that these peptides bind to CaM with similar affinities, the data suggest that having a higher propensity to adopt α-helical structure does not necessarily result in tighter binding, and that the mechanism of CaM binding is very dependent on the nature of the substrate sequence

Role of Sequence and Structure of the Hendra Fusion Protein Fusion Peptide in Membrane Fusion

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion

Functional land management: A framework for managing soil-based ecosystem services for the sustainable intensification of agriculture

peer-reviewedSustainable food production has re-emerged at the top of the global policy agenda, driven by two challenges: (1) the challenge to produce enough food to feed a growing world population and (2) the challenge to make more efficient and prudent use of the world's natural resources. These challenges have led to a societal expectation that the agricultural sector increase productivity, and at the same time provide environmental ‘ecosystem services’ such as the provision of clean water, air, habitats for biodiversity, recycling of nutrients and mitigation against climate change. Whilst the degree to which agriculture can provide individual ecosystem services has been well researched, it is unclear how and to what extent agriculture can meet all expectations relating to environmental sustainability simultaneously, whilst increasing the quantity of food outputs. In this paper, we present a conceptual framework for the quantification of the ‘supply of’ and ‘demand for’ agricultural, soil-based ecosystem services or ‘soil functions’. We use Irish agriculture as a case-study for this framework, using proxy-indicators to determine the demand for individual soil functions, as set by agri-environmental policies, as well as the supply of soil functions, as defined by land use and soil type. We subsequently discuss how this functionality of soils can be managed or incentivised through policy measures, with a view to minimising the divergence between agronomic policies designed to promote increased agricultural production and environmental policy objectives. Finally, we discuss the applicability of this conceptual framework to agriculture and agri-environmental policies at EU level, and the implications for policy makers

\u3csup\u3e1\u3c/sup\u3eH, \u3csup\u3e15\u3c/sup\u3eN, and \u3csup\u3e13\u3c/sup\u3eC Chemical Shift Assignments of the Regulatory Domain of Human Calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13C, 15N and 1H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts

Structural basis for activation of calcineurin by calmodulin

The highly conserved phosphatase calcineurin plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin. Calmodulin binds to a regulatory domain within calcineurin, causing a conformational change that displaces an autoinhibitory domain from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which calmodulin activates calmodulin-dependent protein kinases. Previously published data has hinted that the regulatory domain of calcineurin is intrinsically disordered. In this work we demonstrate that the regulatory domain is unstructured and that it folds upon binding calmodulin, ousting the autoinhibitory domain from the catalytic site. The regulatory domain is 95 residues long, with the autoinhibitory domain attached to its C-terminal end and the 24 residue calmodulin binding region towards the N-terminal end. This is unlike the calmodulin-dependent protein kinases which have calmodulin binding sites and autoinhibitory domains immediately adjacent in sequence. Our data demonstrate that not only does the calmodulin binding region fold, but that an ~25-30 residue region between it and the autoinhibitory domain also folds, resulting in over half of the regulatory domain adopting α-helical structure. This appears to be the first observation of calmodulin inducing folding of this scale outside of its binding site on a target protein

Structural Basis for Activation of Calcineurin by Calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein

Polyglutamine Disruption of the Huntingtin Exon 1 N Terminus Triggers a Complex Aggregation Mechanism

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTTNT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTTNT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTTNT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTTNT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTTNT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntington\u27s disease

Leptospira interrogans Endostatin-Like Outer Membrane Proteins Bind Host Fibronectin, Laminin and Regulators of Complement

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection

Calcineurin

The serine/threonine phosphatase calcineurin acts as a crucial connection between calcium signaling the phosphorylation states of numerous important substrates. These substrates include, but are not limited to, transcription factors, receptors and channels, proteins associated with mitochondria, and proteins associated with microtubules. Calcineurin is activated by increases in intracellular calcium concentrations, a process that requires the calcium sensing protein calmodulin binding to an intrinsically disordered regulatory domain in the phosphatase. Despite having been studied for around four decades, the activation of calcineurin is not fully understood. This review largely focuses on what is known about the activation process and highlights aspects that are currently not understood

Transient disorder: Calcineurin as an example.

How intrinsically disordered proteins and regions evade degradation by cellular machinery evolved to recognize unfolded and misfolded chains remains a vexing question. One potential means by which this can occur is the disorder is transient in nature. That is, the disorder exists just long enough for it to be bound by a partner biomolecule and fold. A review of 30 y of studies of calmodulin’s activation of calcineurin suggests that the regulatory domain of this vital phosphatase is a transiently disordered region. During activation, the regulatory domain progresses from a folded state, to disordered, followed by folding upon being bound by calmodulin. The transient disordered state of this domain is part of a critical intermediate state that facilitates the rapid binding of calmodulin. Building upon “fly-casting” as a means of facilitating partner binding, the mechanism by which calcineurin undergoes activation and subsequent deactivation could be considered “catch and release.