Backbone 1H, 13C and 15N resonance assignment of the ubiquitin specific protease 7 catalytic domain (residues 208-554) in complex with a small molecule ligand.

The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.

Site-specific identification of an aß fibril-heparin interaction site by using solid-state NMR spectroscopy.

At the surface of Aß(1-40) amyloid fibrils that have a threefold molecular symmetry (green in the left picture) a site of interaction of the glycosaminoglycan analogue heparin (blue) was identified. The binding site consists of residues at the N terminus and the turn regions defining the apices of the triangular geometry. Heparin has a lower affinity for Aß(1-40) fibrils having twofold molecular symmetry, thus revealing a remarkable morphological selectivity.

Dimerization of protein G B1 domain at low pH: a conformational switch caused by loss of a single hydrogen bond.

A number of signals in the NMR spectrum of the B1 domain of staphylococcal protein G (GB1) show a chemical shift dependence on the concentration of the protein at pH 3 but not at neutral pH, implying the existence of self-association at low pH. NMR backbone relaxation experiments show that GB1 undergoes a slow conformational exchange at pH 3, which is not seen at higher pH. Analysis of relaxation dispersion experiments yields a self-association constant of 50 mM, and shows that (15)N chemical shift changes in the dimer interface are up to 3 ppm. The shift changes measured from concentration-dependent HSQC spectra and from relaxation dispersion show good consistency. Measurements of chemical shifts as a function of pH show that a hydrogen bond between the sidechains of Asp44 and Gln40 is broken when Asp44 is protonated, and that loss of this hydrogen bond leads to the breaking of the (i, i + 4) backbone helical hydrogen bond from Asp44 HN to Gln40 O, and therefore to a loss of two residues from the C-terminal end of the helix. This weakens the helix structure and facilitates the loss of further helical structure thus permitting dimerization, which is suggested to occur in the same way as observed for the A42F mutant of GB1 (Jee et al., Proteins 2007;71:1420-1431), by formation of an antiparallel beta-sheet between the edge strands 2 in two monomers. The monomer/dimer ratio is thus a finely balanced equilibrium even in the wild type protein.

Pressure-dependent structure changes in barnase on ligand binding reveal intermediate rate fluctuations.

In this work we measured 1H NMR chemical shifts for the ribonuclease barnase at pressures from 3 MPa to 200 MPa, both free and bound to d(CGAC). Shift changes with pressure were used as restraints to determine the change in structure with pressure. Free barnase is compressed by approximately 0.7%. The largest changes are on the ligand-binding face close to Lys-27, which is the recognition site for the cleaved phosphate bond. This part of the protein also contains the buried water molecules. In the presence of d(CGAC), the compressibility is reduced by approximately 70% and the region of structural change is altered: the ligand-binding face is now almost incompressible, whereas changes occur at the opposite face. Because compressibility is proportional to mean square volume fluctuation, we conclude that in free barnase, volume fluctuation is largest close to the active site, but when the inhibitor is bound, the fluctuations become much smaller and are located mainly on the opposite face. The timescale of the fluctuations is nanoseconds to microseconds, consistent with the degree of ordering required for the fluctuations, which are intermediate between rapid uncorrelated side-chain dynamics and slow conformational transitions. The high-pressure technique is therefore useful for characterizing motions on this relatively inaccessible timescale.

Use of quantitative (1)H NMR chemical shift changes for ligand docking into barnase.

(1)H NMR complexation-induced changes in chemical shift (CIS) of HN protons have been used to characterize the complexes of barnase with the deoxyoligonucleotides d(GC) and d(CGAC). Quantitative shift changes are used not only to locate the most probable binding site (using ring-current shifts), but also to determine the orientation of the ligand within the binding site, based on a more complete shift calculation including bond magnetic anisotropies and electric field effects. For both ligands, the guanine is in the same binding site cleft, in the same position as identified in the crystal structure of the d(CGAC) complex. By contrast, a previous X-ray crystal structure of the d(GC) complex showed the ligand in the mouth of the active site, rather than at the guanyl-specific site, implying that the location may be an artifact of the crystallisation process.

Why the Energy Landscape of Barnase Is Hierarchical.

We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s-1, involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10-9-10-6 s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s-1) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-µs timescale.

Interaction of human GTP cyclohydrolase I with its splice variants.

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.

Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets.

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.

Dimerisation of the UBA domain of p62 inhibits ubiquitin binding and regulates NF-kappaB signalling.

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappaB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappaB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K(dim) approximately 4-12 microM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappaB luciferase reporter assays, are associated with reduced NF-kappaB activity compared with wt-p62.

Interaction of human heat shock protein 70 with tumor-associated peptides.

Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (K(d) = 0.58 microm at 25 degrees C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (K(d) = 32 microm) and melanoma antigen recognized by T cells (MART-1) (K(d) = 2.4 microm). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (K(d) = 0.04 microm) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.

Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling.

Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance.

Sequence and structural duality: designing peptides to adopt two stable conformations.

To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intramolecularly disulfide bridged state. Using an iterative process, we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally, for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.