Alexander Grishaev | National Institute of Standards and Technology (original) (raw)

Papers by Alexander Grishaev

ChemPhysChem, May 24, 2016

Acta Crystallographica Section D Structural Biology

Through an expansive international effort that involved data collection on 12 small-angle X-ray s... more Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å−1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å−1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of cons...

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity,... more Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP ve...

TargetingClostridium difficileinfection (CDI) is challenging because treatment options are limite... more TargetingClostridium difficileinfection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed theC. difficiletoxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures foractivated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). ForAsymCDTb, a novel Ca2+bi...

Journal of Biomolecular NMR, 2019

Structural information about ribonucleic acid (RNA) is lagging behind that of proteins, in part d... more Structural information about ribonucleic acid (RNA) is lagging behind that of proteins, in part due to its high charge and conformational variability. Molecular dynamics (MD) has played an important role in describing RNA structure, complementing information from both nuclear magnetic resonance (NMR), or X-ray crystallography. We examine the impact of the choice of the empirical force field for RNA structure refinement using cross-validation against residual dipolar couplings (RDCs) as structural accuracy reporter. Four force fields, representing both the state-of-the art in RNA simulation and the most popular selections in NMR structure determination, are compared for a prototypical A-RNA helix. RNA structural accuracy is also evaluated as a function of both density and nature of input NMR data including RDCs, anisotropic chemical shifts, and distance restraints. Our results show a complex interplay between the experimental restraints and the force fields indicating two best-performing choices: high-fidelity refinement in explicit solvent, and the conformational databasederived potentials. Accuracy of RNA models closely tracks the density of 1-bond C-H RDCs, with other data types having beneficial, but smaller effects. At lower RDC density, or when refining against NOEs only, the two selected force fields are capable of accurately describing RNA helices with little or no experimental RDC data, making them available for the higher order structure assembly or better quantification of the intramolecular dynamics. Unrestrained simulations of simple RNA motifs with state-of-the art MD force fields appear to capture the flexibility inherent in nucleic acids while also maintaining a good agreement with the experimental observables.

Biomolecules, 2019

Folded proteins show a high degree of structural order and undergo (fairly constrained) collectiv... more Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implica...

Science, 2018

Riback et al . (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experi... more Riback et al . (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Förster resonance energy transfer (FRET) experiments. There is thus no “contradiction” between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction.

The Journal of biological chemistry, Jan 25, 2018

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine... more Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg or Mn ions for activity The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1A, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1A D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion...

Journal of molecular biology, Jan 3, 2018

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate ... more Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show ...

Biophysical Journal, 2017

deep in the duplex sequence. Based on these results and our previous studies we propose a possibl... more deep in the duplex sequence. Based on these results and our previous studies we propose a possible model for DNA duplex unwinding by the helicase loaderprimosome-single-strand binding protein complex within the T4 replication system.

Protein science : a publication of the Protein Society, Jan 8, 2017

Using fine-tuned hydrogen bonding criteria, a library of coiled peptide fragments has been genera... more Using fine-tuned hydrogen bonding criteria, a library of coiled peptide fragments has been generated from a large set of high-resolution protein X-ray structures. This library is shown to be an improved representation of ϕ/ψ torsion angles seen in intrinsically disordered proteins (IDPs). The ϕ/ψ torsion angle distribution of the library, on average, provides good agreement with experimentally observed chemical shifts and (3) JHN-Hα coupling constants for a set of five disordered proteins. Inspection of the coil library confirms that nearest-neighbor effects significantly impact the ϕ/ψ distribution of residues in the coil state. Importantly, (3) JHN-Hα coupling constants derived from the nearest-neighbor modulated backbone ϕ distribution in the coil library show improved agreement to experimental values, thereby providing a better way to predict (3) JHN-Hα coupling constants for IDPs, and for identifying locations that deviate from fully random behavior. This article is protected b...

Proceedings of the National Academy of Sciences of the United States of America, Mar 28, 2017

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fra... more Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuat...

Journal of the American Chemical Society, 2016

There has been a long-standing controversy regarding the effect of chemical denaturants on the di... more There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (R g). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of singlemolecule Forster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

ChemPhysChem, 2016

Nucleic-acid-relatede vents in the HIV-1 replication cycle are mediated by nucleocapsid, as mall ... more Nucleic-acid-relatede vents in the HIV-1 replication cycle are mediated by nucleocapsid, as mall protein comprising two zinc knuckles connected by as hort flexible linker and flanked by disordered termini. Combining experimentalN MR residual dipolar couplings, solution X-ray scatteringand protein engineering with ensemble simulated annealing, we obtain aq uantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N-and C-termini that satisfies the experimental restraints, and then validate this ensemble,a sw ella sc haracterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins.D ifferential flexibility within the linker resultsi na symmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at al evel of % 40 %) closely resembles that observed in variousl igand-bound forms,p roviding evidencef or conformational selection and am echanistic link between protein dynamics and function.