Design and characterization of swapped-domain constructs of HIV-1 glycoprotein-41 as receptors for drug discovery.

Four new swapped-domain constructs of the ectodomain of human immunodeficiency virus type 1 glycoprotein-41 (gp41) were prepared. The gp41 ectodomain consists of 50-residue N-heptad repeat (NHR), 36-residue disulfide-bonded loop and 39-residue C-heptad repeat (CHR). It folds into a hairpin structure that forms a trimer along the NHR axis. The swapped-domain proteins feature CHR domains of length 39, 28 or 21 residues preceding a 4-residue loop and a 49- or 50-residue NHR domain. The effect of CHR truncation was to expose increasing lengths of the NHR groove, including the conserved hydrophobic pocket, an important drug target. A novel method for preparing proteins with extended exposed hydrophobic surfaces was demonstrated. Biophysical measurements, including analytical ultracentrifugation and ligand-detected Water-Ligand Observed via Gradient Spectroscopy and (1)H-(15)N-HSQC NMR experiments, were used to confirm that the proteins formed stable trimers in solution with exposed binding surfaces. These proteins could play an important role as receptors in structure-based drug discovery.

Swapped-domain constructs of the glycoprotein-41 ectodomain are potent inhibitors of HIV infection.

The conformational rearrangement of N- and C-heptad repeats (NHR, CHR) of the HIV-1 glycoprotein-41 (gp41) ectodomain into a trimer of hairpins triggers virus-cell fusion by bringing together membrane-spanning N- and C-terminal domains. Peptides derived from the NHR and CHR inhibit fusion by targeting a prehairpin intermediate state of gp41. Typically, peptides derived from the CHR are low nanomolar inhibitors, whereas peptides derived from the NHR are low micromolar inhibitors. Here, we describe the inhibitory activity of swapped-domain gp41 mimics of the form CHR-loop-NHR, which were designed to form reverse hairpin trimers exposing NHR grooves. We observed low nanomolar inhibition of HIV fusion in constructs that possessed the following properties: an extended NHR C-terminus, an exposed conserved hydrophobic pocket on the NHR, high helical content, and trimer stability. Low nanomolar activity was independent of CHR length. CD studies in membrane mimetic dodecylphosphocholine micelles suggested that bioactivity could be related to the ability of the inhibitors to interact with a membrane-associated prehairpin intermediate. The swapped-domain design resolves the problem of unstable and weakly active NHR peptides and suggests a different mechanism of action from that of CHR peptides in inhibition of HIV-1 fusion.

NMR studies on domain diffusion and alignment in modular GB1 repeats.

Modular proteins contain individual domains that are often connected by flexible, unstructured linkers. Using a model system based on the GB1 domain, we constructed tandem repeat proteins and investigated the rotational diffusion and long-range angular ordering behavior of individual domains by measuring NMR relaxation parameters and residual dipolar couplings. Although they display almost identical protein-solvent interfaces, each domain exhibits distinct rotational diffusion and alignment properties. The diffusion tensor anisotropy of the N-terminal domain (NTD) is D(‖)/D(⊥) = 1.5-1.6, similar to that of single-GB1 domains (D(‖)/D(⊥) = 1.6-1.7), whereas the value for the C-terminal domain (CTD) is D(‖)/D(⊥) = 2.0-2.2. In addition, the two domains have different rotational correlation times. These effects are observed for linkers of three to 24 residues, irrespective of linker length. The NTD and CTD also differ in their degree of magnetic alignment, even with a flexible linker of 18 residues, exhibiting D(a) values of 7.7 Hz and 9.7 Hz, respectively. Our results suggest that diffusion differences and long-range influences may persist in modular protein systems, even for systems that have highly flexible linkers and exhibit no domain-domain or domain-linker interactions.

Engineered human antibody constant domains with increased stability.

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.

Dynamics of antibody domains studied by solution NMR.

Information on local dynamics of antibodies is important to evaluate stability, to rationally design variants, and to clarify conformational disorders at the epitope binding sites. Such information may also be useful for improved understanding of antigen recognition. NMR can be used for characterization of local protein dynamics at the atomic level through relaxation measurements. Due to the complexity of the NMR spectra, an extensive use of this method is limited to small protein molecules, for example, antibody domains and some scFv. Here, we describe a protocol that was used to study the dynamics of an antibody domain in solution using NMR. We describe protein preparation for NMR studies, NMR sample optimization, signal assignments, and dynamics experiments.

Periodicity, planarity, and pixel (3P): a program using the intrinsic residual dipolar coupling periodicity-to-peptide plane correlation and phi/psi angles to derive protein backbone structures.

We present a detailed description of a theory and a program called 3P. "3P" stands for periodicity, planarity, and pixel. The 3P program is based on the intrinsic periodic correlations between residual dipolar couplings (RDCs) and in-plane internuclear vectors, and between RDCs and the orientation of peptide planes relative to an alignment tensor. The program extracts accurate rhombic, axial components of the alignment tensor without explicit coordinates, and discrete peptide plane orientations, which are utilized in combination with readily available phi/psi angles to determine the three-dimensional backbone structures of proteins. The 3P program uses one alignment tensor. We demonstrate the utility and robustness of the program, using both experimental and synthetic data sets, which were added with different levels of noise or were incomplete. The program is interfaced to Xplor-NIH via a "3P" module and is available to the public. The limitations and differences between our program and existing methods are also discussed.

The structure of receptor-associated protein (RAP).

The receptor-associated protein (RAP) is a molecular chaperone that binds tightly to certain newly synthesized LDL receptor family members in the endoplasmic reticulum (ER) and facilitates their delivery to the Golgi. We have adopted a divide-and-conquer strategy to solve the structures of the individual domains of RAP using NMR spectroscopy. We present here the newly determined structure of domain 2. Based on this structure and the structures of domains 1 and 3, which were solved previously, we utilized experimental small-angle neutron scattering (SANS) data and a novel simulated annealing protocol to characterize the overall structure of RAP. The results reveal that RAP adopts a unique structural architecture consisting of three independent three-helix bundles that are connected by long and flexible linkers. The flexible linkers and the quasi-repetitive structural architecture may allow RAP to adopt various possible conformations when interacting with the LDL receptors, which are also made of repetitive substructure units.

The structure of free L11 and functional dynamics of L11 in free, L11-rRNA(58 nt) binary and L11-rRNA(58 nt)-thiostrepton ternary complexes.

The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a "reversible switch" in facilitating the coordinated movements associated with EF-G-driven GTP hydrolysis. The reversible switch mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11 complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: first, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a beta-sheet and a 3(10)-helix-turn-helix element in the N terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N terminus, as implied by a decrease of radius of gyration from 18.5 A to 16.2 A. Second, the regions, which undergo large conformation changes, exhibit motions on milliseconds-microseconds or nanoseconds-picoseconds time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 3(10)-helix in L11.

15N solid-state NMR provides a sensitive probe of oxidized flavin reactive sites.

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors.

RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi.

The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.

Determining a helical protein structure using peptide pixels.

The residual dipolar coupling-periodicity planarity correlation makes it possible to determine peptide plane orientations in regular periodic protein secondary structure elements. Each peptide plane orientation represents a "pixel" of protein structure, and is expressed in terms of three angles referred to as tilt, phase, and pitch angles. In this report, we present the novel "3P" (periodicity, planarity, and pixels) method that allows one to determine secondary and tertiary structure of alpha-helical proteins. We demonstrate the 3P method by determining the structure of domain 1 of the receptor-associated protein (RAP) to a backbone accuracy of 1.0 Angstrom using RDCs measured in a single alignment medium, together with a minimal number of NOE distance restraints, using a new Xplor-NIH module.

Periodicity, planarity, residual dipolar coupling, and structures.

The periodic behavior of residual dipolar couplings (RDCs) arising from nucleic acid and protein secondary structures is shown to be more complex and information-rich than previously believed. We have developed a theoretical framework which allows the bond vector orientation of nucleic acids and the peptide plane orientations of protein secondary structures to be extracted from their Dipolar waves. In this article, we focus on utilizing "Dipolar waves" of peptides to extract structure information, and describe in more detail the fundamental principles of the relationship between the periodicities in structure and RDCs, the practical procedure to extract peptide plane orientation information from RDC data, and assessment of errors using Monte-Carlo simulations. We demonstrate the utility of our method for two model alpha-helices, one kinked and one curved, and as well as an irregular beta-strand.

Periodicity in residual dipolar couplings and nucleic acid structures.

The periodicity in nucleic acid duplex structures is shown to be correlated to the periodicity in residual dipolar couplings (RDCs) in the form of an "RDC wave". This "RDC wave" is characteristic of the alignment of the duplex in the magnetic field, and hence fitting of the data allows the duplex global orientation (, Phi) to be extracted. Further, because the "RDC wave" is fit as a data set of a corresponding secondary structure element, the degeneracy problem is greatly reduced. Consequently, with the global orientation (, Phi) determined, local bond vector conformations are defined. The fit is demonstrated in the examples of the imino RDCs of the negative regulator of splicing RNA fragment (NRS23) and for the C1'H1' RDCs of the Dickerson dodecamer.