Kang Chen

Solution NMR Characterizations of Oligomerization and Dynamics of Equine Infectious Anemia Virus Matrix Protein and Its Interaction with PIP2

Budding of retroviruses requires the structural precursor polyprotein, Gag, to target the plasma membrane through its N-terminal matrix (MA) domain. For HIV-1, the interaction between membrane signaling molecule phosphatidylinositol 4,5-diphosphate (PIP2) and MA induces the exposure of myristate and promotes membrane binding. Here we studied oligomerization of the naturally unmyristylated equine infectious anemia virus (EIAV) MA and its interaction with PIP2-C4 primarily using solution NMR spectroscopy. The measured 1H-15N residual dipolar coupling agrees with the atomic coordinates from the EIAV MA crystal structure. The analytical ultracentrifugation results show a dominant population of monomeric EIAV MA at a concentration of 63 microM and 20 degrees C, along with a small trimer and a broad distribution of other oligomers. The monomer-trimer equilibrium model and the quaternary packing of the trimer were further established by the concentration-dependent 15N spin relaxation rates and chemical shifts. Binding of MA to PIP2-C4 was detected by chemical shift mapping (CSM) with an apparent Kd of 182 +/- 56 microM, a value similar to that reported for HIV-1 MA. The PIP2 binding site includes the Loop region between Helix2 and Helix3 in the EIAV MA. CSM and spin relaxation dispersion reveal a coupling of conformational change and submillisecond dynamics, respectively, between the Loop and trimeric Interface Residues due to PIP2 binding. We infer that PIP2 participates in the initial trimer formation of EIAV MA, but more importantly, the concentration effect is dominant in shifting the equilibrium toward trimer, in line with the entropic switch mechanism proposed for myristylated HIV-1 MA.

Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli.

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5‐biphosphate [PI(4,5)P2], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV‐1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P2 is critical for HIV‐1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P2 is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus‐like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3‐phosphate [PI(3)P] with higher affinity than PI(4,5)P2 as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5‐biphosphate [PI(3,5)P2]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P2 from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV‐1, release of EIAV VLPs was not significantly diminished by coexpression with 5‐phosphatase IV, an enzyme that specifically depletes PI(4,5)P2 from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI‐binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag–Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI‐mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

The Use of Residual Dipolar Coupling in Studying Proteins by NMR

The development of residual dipolar coupling (RDC) in protein NMR spectroscopy, over a decade ago, has become a useful and almost routine tool for accurate protein solution structure determination. RDCs provide orientation information of magnetic dipole-dipole interaction vectors within a common reference frame. Its measurement requires a nonisotropic orientation, through a direct or indirect magnetic field alignment, of the protein in solution. There has been recent progress in the developments of alignment methods to allow the measurement of RDC and of methods to analyze the resulting data. In this chapter we briefly go through the mathematical expressions for the RDC and common descriptions of the alignment tensor, which may be represented using either Saupe order or the principal order matrix. Then we review the latest developments in alignment media. In particular we looked at the lipid-compatible media that allow the measurement of RDCs for membrane proteins. Other methods including conservative surface residue mutation have been invented to obtain up to five orthogonal alignment tensors that provide a potential for de novo structure and dynamics study using RDCs exclusively. We then discuss approximations assumed in RDC interpretations and different views on dynamics uncovered from the RDC method. In addition to routine usage of RDCs in refining a single structure, novel applications such as ensemble refinement against RDCs have been implemented to represent protein structure and dynamics in solution. The RDC application also extends to the study of protein-substrate interaction as well as to solving quaternary structure of oligomer in equilibrium with a monomer, opening an avenue for RDCs in high-order protein structure determination.

Water proton spin saturation affects measured protein backbone 15N spin relaxation rates

Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios (s/n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1HN recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (=0.81) than the non-saturated ones (=0.88), indicating such order parameters may be previously underestimated.

Extended model free approach to analyze correlation functions of multidomain proteins in the presence of motional coupling.

Interdomain motion in proteins plays an important role in biomolecular interaction. Its presence also complicates interpretation of many spectroscopy measurements. Nuclear magnetic resonance (NMR) study of domain dynamics relies on knowledge of its rotational correlation function. The extended model free (EMF) approach has been implemented to analyze coupled domain and overall motions for calmodulin, a dual-domain protein; however, the validity of EMF treatment in coupled motion has not been tested. We performed stochastic simulations on a dual-vector system employing two simple restraints to drive hydrodynamics and domain coupling: (1) both unitary vectors diffuse randomly on the surface of a sphere and (2) vectors are correlated through user-defined intervector potential. The resulting correlation curve can be adequately fit with either a single- or double-exponential decay function. The latter is consistent with the EMF treatment. The derived order parameters S (2) range from about 0.4 to 1, while the motion separation, the ratio of overall and domain motion time scales (tau m/tau s), ranges from 1 to 4. A complete overlap between time scales occurs when S (2) is less than 0.4, and the correlation function effectively behaves as a single-exponential. The S (2) values are consistent with theoretical predictions from the given potential function, differing by no more than 0.03, suggesting EMF to be a generally valid approach. In addition, from the dependence of S (2) on tau m/tau s obtained from simulation, we found a cosine potential, favoring extended conformers, as opposed to the normally assumed cone potential, reached a better agreement to experimental data.

The Maturational Refolding of the β-Hairpin Motif of Equine Infectious Anemia Virus Capsid Protein Extends Its Helix α1 at Capsid Assembly Locus

Background: The function of the maturational refolded N-terminal β-hairpin in retroviral capsid remains unknown. Results: Folding the β-hairpin of equine infectious anemia virus (EIAV) capsid extends its downstream helix α1 at the N terminus, which forms the oligomerization core of retroviral capsids. Conclusion: The β-hairpin structures helix α1, which could be necessary for capsid assembly. Significance: Solution NMR revealed the function of the puzzling β-hairpin motif in retroviral capsid. A retroviral capsid (CA) protein consists of two helical domains, CAN and CAC, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr20 to Pro17. This coil to helix transition involves the conserved sequence of Thr16-Pro17-Arg18 (Ser16-Pro17-Arg18 in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CAN oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg18, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CAN oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CAN domain oligomerization, independent to the faster EIAV-CAC domain dimerization.

Determining interdomain structure and dynamics of a retroviral capsid protein in the presence of oligomerization: implication for structural transition in capsid assembly.

Capsid (CA) proteins from all retroviruses, including HIV-1, are structurally homologous dual-domain helical proteins. They form a capsid lattice composed of unitary symmetric CA hexamers. X-ray crystallography has shown that within each hexamer a monomeric CA adopts a single conformation, where most helices are parallel to the symmetry axis. In solution, large differences in averaged NMR spin relaxation rates for the two domains were observed, suggesting they are dynamically independent. One relevant question for the capsid assembly remains: whether the interdomain conformer within a hexamer unit needs to be induced or pre-exists within the conformational space of a monomeric CA. The latter seems more consistent with the relaxation data. However, possible CA protein oligomerization and the structure of each domain will affect relaxation measurements and data interpretation. This study, using CA proteins from equine infectious anemia virus (EIAV) as an example, demonstrates a linear extrapolation approach to obtain backbone (15)N spin relaxation time ratios T1/T2 for a monomeric EIAV-CA in the presence of oligomerization equilibrium. The interdomain motion turns out to be limited. The large difference in the domain averaged for a CA monomer is a consequence of the orthogonal distributions of helices in the two domains. The new monomeric interdomain conformation in solution is significantly different from that in CA hexamer. Therefore, if capsid assembly follows a nucleation-propagation process, the interdomain conformational change might be a key step during the nucleation, as the configuration in hexagonal assembly is never formed by diffusion of its two domains in solution.

Chapter 8:Solution NMR Spectroscopy in Characterizing Structure, Dynamics and Intermolecular Interactions of Retroviral Structural Proteins

Solution nuclear magnetic resonance (NMR) spectroscopy has been an important tool in modern structural biology owing to the development of isotope labeling methods, triple-resonance pulse sequence techniques, specialized cryogenic probes and higher magnetic field strengths. NMR spectroscopy allows t...