Shangjin Sun

Enhanced Sensitivity by Nonuniform Sampling Enables Multidimensional MAS NMR Spectroscopy of Protein Assemblies

We report dramatic sensitivity enhancements in multidimensional MAS NMR spectra by the use of nonuniform sampling (NUS) and introduce maximum entropy interpolation (MINT) processing that assures the linearity between the time and frequency domains of the NUS acquired data sets. A systematic analysis of sensitivity and resolution in 2D and 3D NUS spectra reveals that with NUS, at least 1.5- to 2-fold sensitivity enhancement can be attained in each indirect dimension without compromising the spectral resolution. These enhancements are similar to or higher than those attained by the newest-generation commercial cryogenic probes. We explore the benefits of this NUS/MaxEnt approach in proteins and protein assemblies using 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) Escherichia coli thioredoxin reassembly. We demonstrate that in thioredoxin reassembly, NUS permits acquisition of high-quality 3D-NCACX spectra, which are inaccessible with conventional sampling due to prohibitively long experiment times. Of critical importance, issues that hinder NUS-based SNR enhancement in 3D-NMR of liquids are mitigated in the study of solid samples in which theoretical enhancements on the order of 3-4 fold are accessible by compounding the NUS-based SNR enhancement of each indirect dimension. NUS/MINT is anticipated to be widely applicable and advantageous for multidimensional heteronuclear MAS NMR spectroscopy of proteins, protein assemblies, and other biological systems.

Solid-state and solution NMR studies of the CAP-Gly domain of mammalian dynactin and its interaction with microtubules.

Microtubules (MTs) and microtubule binding proteins (MTBPs) play fundamental physiological roles including vesicle and organelle transport, cell motility, and cell division. Despite the importance of the MT/MTBP assemblies, there remains virtually no structural or dynamic information about their interaction at the atomic level due to the inherent insolubility and lack of long-range order of MTs. In this study, we present a combined magic angle spinning solid-state and solution NMR study of the MTBP CAP-Gly domain of mammalian dynactin and its interaction with paclitaxel-stabilized microtubules. We report resonance assignments and secondary structure analysis of the free CAP-Gly in solution and in the solid state by a combination of two- and three-dimensional homo- and heteronuclear correlation spectra. In solution, binding of CAP-Gly to microtubules is accompanied by the broadening of the majority of the peaks in HSQC spectra except for the residues at the termini, precluding further structural analysis of the CAP-Gly/microtubule complexes. In the solid state, DARR spectra of free CAP-Gly and its complex with microtubules display well-resolved lines, permitting residue-specific resonance assignments. Interestingly, a number of chemical shifts in the solid-state DARR spectra of the CAP-Gly/microtubule complex are perturbed compared to those of the free CAP-Gly, suggesting that conformational changes occur in the protein upon binding to the microtubules. These results indicate that CAP-Gly/microtubule assemblies are amenable to detailed structural characterization by magic angle spinning NMR spectroscopy and that solid-state NMR is a viable technique to study MT/protein interactions in general.

Biochemical and structural characterization of the Pak1-LC8 interaction.

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser88 inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser88. Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.

Spin Diffusion Driven by R-Symmetry Sequences: Applications to Homonuclear Correlation Spectroscopy in MAS NMR of Biological and Organic Solids

We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation spectra both in model compounds and in proteins. Under these conditions, individual R2(n)(v) display different polarization transfer efficiency dependencies on isotropic chemical shift differences: R2(2)(1) recouples efficiently both small and large chemical shift differences (in proteins these correspond to aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations, respectively), while R2(1)(1) and R2(2)(2) exhibit the maximum recoupling efficiency for the aliphatic-to-aliphatic or carbonyl-to-aliphatic correlations, respectively. At moderate MAS frequencies (10-20 kHz), all R2(n)(v) sequences introduced in this work display similar transfer efficiencies, and their performance is very similar to that of PDSD and DARR. Polarization transfer dynamics and chemical shift dependencies of these R2-driven spin diffusion (RDSD) schemes are experimentally evaluated and investigated by numerical simulations for [U-(13)C,(15)N]-alanine and the [U-(13)C,(15)N] N-formyl-Met-Leu-Phe (MLF) tripeptide. Further applications of this approach are illustrated for several proteins: spherical assemblies of HIV-1 U-(13)C,(15)N CA protein, U-(13)C,(15)N-enriched dynein light chain DLC8, and sparsely (13)C/uniformly (15)N enriched CAP-Gly domain of dynactin. Due to the excellent performance and ease of implementation, the presented R2(n)(v) symmetry sequences are expected to be of wide applicability in studies of proteins and protein assemblies as well as other organic solids by MAS NMR spectroscopy.

A Time-Saving Strategy for MAS NMR Spectroscopy by Combining Nonuniform Sampling and Paramagnetic Relaxation Assisted Condensed Data Collection

We present a time-saving strategy for acquiring 3D magic angle spinning NMR spectra for chemical shift assignments in proteins and protein assemblies in the solid state. By simultaneous application of nonuniform sampling (NUS) and paramagnetic-relaxation-assisted condensed data collection (PACC), we can attain 16-fold time reduction in the 3D experiments without sacrificing the signal-to-noise ratio or the resolution. We demonstrate that with appropriate concentration of paramagnetic dopant introduced into the sample the overwhelming majority of chemical shifts are not perturbed, with the exception of a limited number of shifts corresponding to residues located at the surface of the protein, which exhibit small perturbations. This approach enables multidimensional MAS spectroscopy in samples of intrinsically low sensitivity and/or high spectral congestion where traditional experiments fail, and is especially beneficial for structural and dynamics studies of large proteins and protein assemblies.

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10–44, is sufficient for binding p150Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

Solid-state NMR spectroscopy of protein complexes.

Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble, and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility, or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR-based methods to study several protein complexes. In this chapter, we discuss the general SSNMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies, and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection, and data analysis.

Disease-Associated Mutations in the p150Glued Subunit Destabilize the CAP-gly Domain

Point mutations within the CAP-gly domain of the p150(Glued) subunit of the dynactin complex have been identified in patients with distal spinal bulbar muscular atrophy (dSBMA) and Perrys syndrome. Herein, we show by CD and NMR experiments that each mutated CAP-gly domain is folded but less stable than the wild-type (WT) domain. We also demonstrate that the domains harboring these mutations bind to microtubules but fail to bind to EB1. These data indicate that these disease-associated, point mutations affect the stability of this domain and inhibit their interaction with EB1 but do not inhibit their interaction with microtubules.