How a highly acidic SH3 domain folds in the absence of its charged peptide target.

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye-Huckel screening and a nonspecific territorial ion-binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short-range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years.

How a highly acidic SH3 domain folds in the absence of its charged peptide target.

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net-charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the 60-residue yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations according to the Debye-Huckel limiting law. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occurs in the transition state. After the transition state formation, modest yet favorable short-range salt-bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over one billion years. Statement for broader audience: Some protein domains are highly charged because they are adapted to bind oppositely charged proteins and nucleic acids. However, it is unknown how these highly charged domains fold as during folding there will be significant repulsion between like-charges. We investigate how one of these highly charged domains folds in the presence of salt, which can screen the charge repulsion and make folding easier, allowing us to understand how folding occurs despite the protein’s high charge. Supplementary material: Supplementary material document containing additional details on protein expression methods, thermodynamics and kinetics equations, and the effect of urea on electrostatic interactions, as well as 4 supplemental figures and 4 supplemental data tables. ( Supplementary_Material.docx ), 15 pages Supplemental excel file containing covariation data across AbpSH3 orthologs ( FileS1.xlsx ).

The Impacts of the Molecular Education and Research Consortium in Undergraduate Computational Chemistry on the Careers of Women in Computational Chemistry.

The Molecular Education and Research Consortium in Undergraduate Computational Chemistry (MERCURY) has supported a diverse group of faculty and students for over 20 years by providing computational resources as well as networking opportunities and professional support. The consortium comprises 38 faculty (42% women) at 34 different institutions, who have trained nearly 900 undergraduate students, more than two-thirds of whom identify as women and one-quarter identify as students of color. MERCURY provides a model for the support necessary for faculty to achieve professional advancement and career satisfaction. The range of experiences and expertise of the consortium members provides excellent networking opportunities that allow MERCURY faculty to support each other's teaching, research, and service needs, including generating meaningful scientific advancements and outcomes with undergraduate researchers as well as being leaders at the departmental, institutional, and national levels. While all MERCURY faculty benefit from these supports, the disproportionate number of women in the consortium, relative to their representation in computational sciences generally, produces a sizable impact on advancing women in the computational sciences. In this report, the women of MERCURY share how the consortium has benefited their careers and the careers of their students.

An Unbound Proline-Rich Signaling Peptide Frequently Samples Cis Conformations in Gaussian Accelerated Molecular Dynamics Simulations.

Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis, a robust methodology to accurately account for these conformations in the overall ensemble is crucial. Observing the cis conformations of proline in a disordered sequence is challenging both experimentally and computationally. Nitrogen-hydrogen NMR spectroscopy cannot directly observe proline residues, which lack an amide bond, and computational methods struggle to overcome the large kinetic barrier between the cis and trans states, since isomerization usually occurs on the order of seconds. In the current work, Gaussian accelerated molecular dynamics was used to overcome this free energy barrier and simulate proline isomerization in a tetrapeptide (KPTP) and in the 12-residue proline-rich SH3 binding peptide, ArkA. We found that Gaussian accelerated molecular dynamics, when combined with a lowered peptide bond dihedral angle potential energy barrier (15 kcal/mol), allowed sufficient sampling of the proline cis and trans states on a microsecond timescale. All ArkA prolines spend a significant fraction of time in cis, leading to a more compact ensemble with less polyproline II helix structure than an ArkA ensemble with all peptide bonds in trans. The ensemble containing cis prolines also matches more closely to in vitro circular dichroism data than the all-trans ensemble. The ability of the ArkA prolines to isomerize likely affects the peptide's ability to bind its partner SH3 domain, and should be studied further. This is the first molecular dynamics simulation study of proline isomerization in a biologically relevant proline-rich sequence that we know of, and a similar protocol could be applied to study multi-proline isomerization in other proline-containing proteins to improve conformational diversity and agreement with in vitro data.

A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway.

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general.

Conformational Dynamics of the HIV-Vif Protein Complex.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-ß. A five-protein complex containing the substrate receptor (Vif, CBF-ß, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the µs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Non-degradative Ubiquitination of Protein Kinases.

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ß-amyloid peptides from E. coli for NMR-based structural analysis.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized ß-amyloid peptides with Aß42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aß42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aß42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aß42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aß42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aß42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aß42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aß42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aß42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aß42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aß42 peptide as well as its other variants including any Aß42 peptide mutants.

Comparison of structure determination methods for intrinsically disordered amyloid-ß peptides.

Intrinsically disordered proteins (IDPs) represent a new frontier in structural biology since the primary characteristic of IDPs is that structures need to be characterized as diverse ensembles of conformational substates. We compare two general but very different ways of combining NMR spectroscopy with theoretical methods to derive structural ensembles for the disease IDPs amyloid-ß 1-40 and amyloid-ß 1-42, which are associated with Alzheimer's Disease. We analyze the performance of de novo molecular dynamics and knowledge-based approaches for generating structural ensembles by assessing their ability to reproduce a range of NMR experimental observables. In addition to the comparison of computational methods, we also evaluate the relative value of different types of NMR data for refining or validating the IDP structural ensembles for these important disease peptides.

Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease.

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aß known as "globulomers", which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aß oligomers yielded signs of the disease, including cell culture age-dependent binding of Aß and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aß-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease.

Differences in ß-strand populations of monomeric Aß40 and Aß42.

Using homonuclear (1)H NOESY spectra, with chemical shifts, (3)JH(N)H(α) scalar couplings, residual dipolar couplings, and (1)H-(15)N NOEs, we have optimized and validated the conformational ensembles of the amyloid-ß 1-40 (Aß40) and amyloid-ß 1-42 (Aß42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel ß-strands. The most significant difference in the structural ensembles of the two peptides is the type of ß-hairpins and ß-strands they populate. We find that Aß42 forms a major antiparallel ß-hairpin involving the central hydrophobic cluster residues (16-21) with residues 29-36, compatible with known amyloid fibril forming regions, whereas Aß40 forms an alternative but less populated antiparallel ß-hairpin between the central hydrophobic cluster and residues 9-13, that sometimes forms a ß-sheet by association with residues 35-37. Furthermore, we show that the two additional C-terminal residues of Aß42, in particular Ile-41, directly control the differences in the ß-strand content found between the Aß40 and Aß42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aß40 and Aß42 consistent with available information that produce a plausible molecular basis for why Aß42 exhibits greater fibrillization rates than Aß40.

Homogeneous and heterogeneous tertiary structure ensembles of amyloid-ß peptides.

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-ß 1-42 (Aß42), the primary peptide associated with Alzheimer's disease, and fragments such as Aß21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aß42 and Aß21-30 from the perspective of their classification as IDPs. Unlike the Aß21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aß42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.

The influence of protein dynamics on the success of computational enzyme design.

We characterize the molecular dynamics of a previously described computational de novo designed enzyme optimized to perform a multistep retrol-aldol reaction when engineered into a TIM barrel protein scaffold. The molecular dynamics simulations show that the protein dynamics under physiological conditions of temperature and aqueous environment distorts the designed geometric factors of the substrate-enzyme reaction intermediates, such that catalysis is limited by the primary retrol-aldol step of proton abstraction from the covalently bound substrate and its interactions with a histidine-aspartate dyad. These results emphasize that computational enzyme designs will benefit from considerations of dynamical fluctuations when optimizing active site geometries.