Structure of a low-population intermediate state in the release of an enzyme product.

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product

The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/E019927/1 to C.M.D., C.V.R., and J.R.K.), the Medical Research Council (E.D.G. and C.M.D.), the Belgian Program of Interuniversity Attraction Poles administered by the Federal Office for Scientific Technical and Cultural Affairs (PAI numbers P6/19 and P7144 to C.M.D. and M.D.), the European Union’s Sixth Framework Program (LSHM-CT-2006-037525 to C.M.D. and M.D.), and Programme grants from the Wellcome Trust and the Leverhulme Trust (C.M.D.). It was also supported by a Korean Government Scholarship for Overseas Studies (M.A.), the Winston Churchill Foundation (C.L.H.), and Boerhinger Ingleheim funds (A.D.). The NMR facility at the Department of Chemistry, University of Cambridge, is supported in part by an EPSRC Core Capability grant (EP/K039520/1)

Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release

Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. © 2013 Aprile et al.FAA was recipient of a graduate fellowship from the Italian Ministry of Education, University and Research. AD is grateful for support from Murray Edwards College, Cambridge, through a Junior Research Fellowship. FS is a Sir Henry Wellcome Fellow. CR acknowledges financial support by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', through ISCIII with cofunding by FEDER (CP10/00527). JLPB is a Royal Society University Research Fellow. FAA and PT are grateful for support from Regione Lombardia (NEDD and >Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi>). CMD acknowledges support from BBSRC (BB/E019927/1), the Wellcome Trust (094425/Z/10/Z), the European Commission (project LSHM-CT-2006-037525). NC acknowledges support from Human Frontiers Science Program (HFSP) through a Long-term Fellowship (LT000795/2009).Peer Reviewe

Disulfide bonds reduce the toxicity of the amyloid fibrils formed by an extracellular protein

In a stable condition: Disulfide bonds stabilize folded proteins primarily by decreasing the entropic cost of folding. Such cross-links also reduce toxic aggregation by favoring the formation of highly structured amyloid fibrils (see picture). It is suggested that disulfide bonds in extracellular proteins were selected by evolutionary pressures because they decrease the propensity to form toxic aggregates. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Application of Lysine-specific Labeling to Detect Transient Interactions Present During Human Lysozyme Amyloid Fibril Formation.

Populating transient and partially unfolded species is a crucial step in the formation and accumulation of amyloid fibrils formed from pathogenic variants of human lysozyme linked with a rare but fatal hereditary systemic amyloidosis. The partially unfolded species possess an unstructured β-domain and C-helix with the rest of the α-domain remaining native-like. Here we use paramagnetic relaxation enhancement (PRE) measured by NMR spectroscopy to study the transient intermolecular interactions between such intermediate species. Nitroxide spin labels, introduced specifically at three individual lysine residues, generate distinct PRE profiles, indicating the presence of intermolecular interactions between residues within the unfolded β-domain. This study describes the applicability to PRE NMR measurements of selective lysine labeling, at different sites within a protein, as an alternative to the introduction of spin labels via engineered cysteine residues. These results reveal the importance of the β-sheet region of lysozyme for initiating self-assembly into amyloid fibrils

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.This work was supported by grants BIO2009-09562, CSD2009-00088 from the Spanish Ministry of Science and Innovation, and FEDER Funds (JMS-R)

Binding Free Energy Landscape of Domain-Peptide Interactions

Peptide recognition domains (PRDs) are ubiquitous protein domains which mediate large numbers of protein interactions in the cell. How these PRDs are able to recognize peptide sequences in a rapid and specific manner is incompletely understood. We explore the peptide binding process of PDZ domains, a large PRD family, from an equilibrium perspective using an all-atom Monte Carlo (MC) approach. Our focus is two different PDZ domains representing two major PDZ classes, I and II. For both domains, a binding free energy surface with a strong bias toward the native bound state is found. Moreover, both domains exhibit a binding process in which the peptides are mostly either bound at the PDZ binding pocket or else interact little with the domain surface. Consistent with this, various binding observables show a temperature dependence well described by a simple two-state model. We also find important differences in the details between the two domains. While both domains exhibit well-defined binding free energy barriers, the class I barrier is significantly weaker than the one for class II. To probe this issue further, we apply our method to a PDZ domain with dual specificity for class I and II peptides, and find an analogous difference in their binding free energy barriers. Lastly, we perform a large number of fixed-temperature MC kinetics trajectories under binding conditions. These trajectories reveal significantly slower binding dynamics for the class II domain relative to class I. Our combined results are consistent with a binding mechanism in which the peptide C terminal residue binds in an initial, rate-limiting step

Local Cooperativity in an Amyloidogenic State of Human Lysozyme Observed at Atomic Resolution

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein