NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.

NMR solution structure of the human prion protein

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90–230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered “tail.” The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–228 and a short anti-parallel β-sheet comprising the residues 128–131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219–228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219–226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

NMR structures of three single-residue variants of the human prion protein

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121–230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrPC) that is formed by the C-terminal part of helix 3, with residues 218–230, and a loop of residues 166–172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized “protein X,” and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121–230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized “conformational markers” could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166–172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166–172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O2 binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both α- and β-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring 1H and 15N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

Elucidating the mechanism of familial amyloidosis– Finnish type: NMR studies of human gelsolin domain 2

Familial amyloidosis–Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between β-strand 4 and α-helix 1 and within the loop region following β-strand 5. Chemical shift differences are noted along the face of α-helix 1 that packs onto the β-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. {1H-15N} nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of β-sheets in Alzheimer's β-amyloid fibrils

Senile plaques associated with Alzheimer's disease contain deposits of fibrils formed by 39- to 43-residue β-amyloid peptides with possible neurotoxic effects. X-ray diffraction measurements on oriented fibril bundles have indicated an extended β-sheet structure for Alzheimer's β-amyloid fibrils and other amyloid fibrils, but the supramolecular organization of the β-sheets and other structural details are not well established because of the intrinsically noncrystalline, insoluble nature of amyloid fibrils. Here we report solid-state NMR measurements, using a multiple quantum (MQ) 13C NMR technique, that probe the β-sheet organization in fibrils formed by the full-length, 40-residue β-amyloid peptide (Aβ1–40). Although an antiparallel β-sheet organization often is assumed and is invoked in recent structural models for full-length β-amyloid fibrils, the MQNMR data indicate an in-register, parallel organization. This work provides site-specific, atomic-level structural constraints on full-length β-amyloid fibrils and applies MQNMR to a significant problem in structural biology.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Using O2 to probe membrane immersion depth by 19F NMR

A fluorinated detergent, CF3(CF2)5C2H4-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by 19F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on 19F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (R1) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O2 at least 100-fold (i.e., PO2 greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (R120bar − R10) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(N,N-dimethyl-N-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in 19F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by 19F NMR.

High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids

The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.

Solution structure and dynamics of GCN4 cognate DNA: NMR investigations

A 12 bp long GCN4-binding, self-complementary duplex DNA d(CATGACGTCATG)2 has been investigated by NMR spectroscopy to study the structure and dynamics of the molecule in aqueous solution. The NMR structure of the DNA obtained using simulated annealing and iterative relaxation matrix calculations compares quite closely with the X-ray structure of ATF/CREB DNA in complex with GCN4 protein (DNA-binding domain). The DNA is also seen to be curved in the free state and this has a significant bearing on recognition by the protein. The dynamic characteristics of the molecule have been studied by 13C relaxation measurements at natural abundance. A correlation has been observed between sequence-dependent dynamics and recognition by GCN4 protein.

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.