Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts

In the chloroplast, Calvin–Benson–Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23–C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66–C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66–C75 pair than for the C23–C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment imposed in the light via the electrons from the photosystems. In the dark these enzymes are inhibited, and this regulation is mainly mediated via oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges are simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment that is, in cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair compared to the C23-C31 pair. Finally, the redox mid-potentials for the two cysteine pairs differ and are similar to those found for phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, that we relate to the regulatory role of CP12.

Sequence-Dependent Correlated Segments in the Intrinsically Disordered Region of ChiZ

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5–10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose the correlated segment as a defining feature for the conformations and dynamics of IDPs.

Sequence-dependent correlated segments in the intrinsically disordered region of ChiZ

Intrinsically disordered proteins (IDPs) account for a significant fraction of any proteome and are central to numerous cellular functions. Yet how sequences of IDPs code for their conformational dynamics is poorly understood. Here we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. This IDP is the N-terminal fragment (residues 1-64) of the transmembrane protein ChiZ, a component of the cell division machinery in Mycobacterium tuberculosis. Its N-half contains most of the prolines and all of the anionic residues while the C-half most of the glycines and cationic residues. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11-29) emerge as “correlated segments”, identified by frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11-14 and 23-28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent: segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5-10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose correlated segment as a defining feature for the conformation and dynamics of IDPs.

Alpha protons as NMR probes in deuterated proteins

We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα–Hα and Hα–HN contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with HN–HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131–294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine–proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

Structural Basis for the Procofactor to Cofactor Transition in Human Factor V

Factor V, the inactive precursor to factor Va, has a domain organization of A1-A2-B-A3-C1-C2. Factor Va is formed by the proteolytic excision of the central B domain, which resolves the molecule into a heterodimer (A1-A2/A3-C1-C2). Removal of the B domain enables the cofactor to engage factor Xa on phosphatidylserine-containing membranes, assemble prothrombinase and greatly enhance the rate of thrombin formation. Recent studies have shown a key role for a basic region (BR), which lies approximately in the center of the B domain, in enforcing procofactor properties in human factor V (hFV). Exogenously added recombinant BR fragments can bind with high affinity to a cofactor-like variant of human hFV (hFVDT), in which a large central portion of the B domain has been deleted, interfere with Xa binding and restore procofactor-like properties. Biochemical evidence suggests that BR binding results from its interaction with an acidic region (AR2) at the C terminus of the B domain and likely also an acidic sequence (AR1) at the C terminus of the A2 domain. Our recent crystal structure of hFVDT provided the first structural evidence that AR1 and AR2, ~800 residues apart in the primary structure of hFV, are positioned adjacent to each other and could plausibly form an extended surface for high affinity BR binding to reconstitute a tripartite procofactor-regulatory region (AR1/BR/AR2). However, the lack of BR in hFVDT precluded independent structural verification of this possibility. In a computational approach, we created a molecular model for the 58 residue BR peptide. The top scoring three-dimensional models of the 58 residue BR peptide showed a helix-loop arrangement, contrary to the general belief that the B domain lacks structured regions. The best scoring BR peptide model was used for ab initio docking studies using the crystal structure of hFVDT to predict possible binding sites using PIPER and ClusPro. The most highly represented and statistically probable solutions showed the BR peptide in intimate contact with juxtaposed surfaces provided by AR1 and AR2. Interestingly, the docked BR peptide contacted regions in AR1 and on the A2 domain implicated in FXa binding in the structure of Pseudonaja textilis FV bound to snake venom factor X. Computational predictions were tested using hydrogen-deuterium exchange detected by protein fragmentation and mass spectroscopy (HDX). Proteolytic fragmentation of hFVDT and fragment detection by LC-MS was optimized to cover >95% of its 1514 residues with an average redundancy of 4.27 peptides/residue. Only 4 or 5 segments of ~10-15 residue length were not covered. Addition of the BR peptide had minor effects on amide proton exchange over the bulk of the molecule. However, BR peptide binding was accompanied by reductions in amide proton exchange rates of ~7-30-fold in immediately adjacent regions of hFVDT corresponding to sequences within A2 (626-634), AR1 (658-695), AR2 (872-881) and A3 (983-995). BR peptide binding to hFVDT is accompanied by perturbations in these spatially adjacent regions covering a small fraction of the surface area at approximately the 3 o'clock position with the molecule in the standard orientation. The marked agreement between the HDX findings and the computational docking studies supports our proposal that the BR engages an extended surface contributed by AR1 and AR2 to form a tripartite procofactor-regulatory region. The interaction of BR with AR1 and a small region in A2, both implicated in binding Xa, potentially explains how the BR might restrict Xa binding to the procofactor. Destabilization of BR binding by proteolysis at the C terminus of AR2 is envisioned to result in cofactor formation by releasing the BR and revealing sites responsible for binding Xa. Our findings provide a structural explanation for the long standing puzzle of factor V activation and pave the way for further definition of mechanistic details of procofactor and cofactor function. They have implications for how interactions with TFPIα through the basic region at its C-terminus might regulate FV(a). They also reveal previously unanticipated strategies to modulate functions of hFV and hFVa for therapeutic gain. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; Bayer: Consultancy; Novo Nordisk: Research Funding; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Krishnaswamy:Portola: Research Funding; Janssen: Consultancy, Research Funding.