Joanna K. Krueger

Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling.

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the αD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM3CSK4 mediated NF-κB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.

Cutting Edge: CD4 Is the Receptor for the Tick Saliva Immunosuppressor, Salp15

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.

Antiviral peptides targeting the west nile virus envelope protein.

ABSTRACT West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

Gelsolin regulates the dynamic assembly and disassembly of the actin-based cytoskeleton in non-muscle cells and clears the circulation of filaments released following cell death. Gelsolin is a six-domain (G1-G6) protein activated by calcium via a multi-step process that involves unfolding from a compact form to a more open form in which the three actin-binding sites (on the G1, G2, and G4 subdomains) become exposed. To follow the global structural changes that accompany calcium activation of gelsolin, small-angle x-ray scattering (SAXS) data were collected for full-length human plasma gelsolin at nanomolar to millimolar concentrations of free Ca2+. Analysis of these data showed that, upon increasing free Ca2+ levels, the radius of gyration (Rg) increased nearly 12 Å, from 31.1 ± 0.3 to 43 ± 2 Å, and the maximum linear dimension (Dmax) of the gelsolin molecule increased 55 Å, from 100 to 155Å. Structural reconstruction of gelsolin from these data provided a striking visual tracking of the gradual Ca2+-induced opening of the gelsolin molecule and highlighted the critical role played by the flexible linkers between homologous domains. The tightly packed architecture of calcium-free gelsolin, seen from both SAXS and x-ray crystallographic models, is already partially opened up in as low as 0.5 nm Ca2+. Our data confirm that, although the molecule springs open from 0 to 1 μm free Ca2+, even higher calcium concentrations help to stabilize a more open structure, with increases in Rg and Dmax of ∼2 and ∼15 Å, respectively. At these higher calcium levels, the SAXS-based models provide a molecular shape that is compatible with that of the crystal structures solved for Ca2+/gelsolin C-terminal and N-terminal halves ± monomeric G-actin. Placement of these crystal structures within the boundaries of the SAXS-based model suggests a movement of the G1/G2 subunits that would be required upon binding to actin.

Activation of myosin light chain kinase requires translocation of bound calmodulin.

A novel translocation step is inferred from structural studies of the interactions between the intracellular calcium receptor protein calmodulin (CaM) and one of its regulatory targets. A mutant of CaM missing residues 2–8 (ΔNCaM) binds skeletal muscle myosin light chain kinase with high affinity but fails to activate catalysis. Small angle x-ray scattering data reveal that ΔNCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM residues 2–8 appear to facilitate movement of bound CaM away from the vicinity of the catalytic cleft.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-Independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 Å resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 Å root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 Å) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides α-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Conformational Rearrangement within the Soluble Domains of the CD4 Receptor Is Ligand-specific

Ligand binding induces shape changes within the four modular ectodomains (D1–D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed that both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2–D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2–D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.

Dehaloperoxidase-hemoglobin from Amphitrite ornata is primarily a monomer in solution.

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.

Efforts to Implement a PhD degree program in “Nanoscale Science” at UNC Charlotte

UNC Charlotte is a young and growing research university. Most of the Ph.D. programs on our campus have been designed to be interdisciplinary. This strategic choice was made for both economic and pedagogical reasons. At the heart of the drive for interdisciplinary degree programs is the recognition that a lack of educational diversity at the Ph.D. level is limiting for new graduates in today’s research and discovery landscape. This need for educational diversity is even more acute in the sciences. We need more chemists that know more physics, and we need more physicists that know more biology, and we need more engineers that understand matter at a molecular scale. To this end, faculty in the departments of chemistry, optical sciences, mechanical engineering, and electrical engineering have designed and are implementing a new interdisciplinary Ph.D. degree in “Nanoscale Science”. We do not believe that a length scale can institute a philosophy of science. However, research involving nanoscale materials and phenomena do require an educational perspective far broader than traditional academic disciplines currently offer. The question is how to deliver a broad graduate education that enables each student to reach an expertise required for the Ph.D. This is the question that has driven our pedagogical development of this Nanoscale science program. The overall structure of this program will be described and compared to other current efforts in Nanoscale graduate education throughout the United States. Various novel features will be discussed, with the hope for critical feedback and discussion. Details of the educational opportunities we have designed and the method of assessment we will employ will be presented. Background The Need for Ph.D. Programs in Nanoscale Science The National Science Foundation estimates that nanotechnology will be a

Regular articleThe assembly of immunoglobulin-like modules in titin: implications for muscle elasticity1

1 trillion global industry by 2010-2015. This will require about 2 million workers in the field of nanoscale science and engineering, of which 0.8-0.9 million will be in the United Mater. Res. Soc. Symp. Proc. Vol. 931