Solution structure of human apolipoprotein(a) kringle IV type 6.

The structure of apo(a) KIVT6 was investigated by two- and three-dimensional homo- and heteronuclear NMR spectroscopy. The solution structure of apo(a) KIVT6 contains only a small amount of regular secondary structure elements, comprising a short piece of antiparallel beta-sheet formed by residues Trp62-Tyr64 and Trp72-Tyr74, a short piece of parallel beta-sheet formed by the residues Cys1-Tyr2 and Thr78-Gln79, and a small 3(10)-helix within residues Thr38-Tyr40. The backbone as well as the side chains are arranged in a way similar to those of apo(a) KIVT7, apo(a) KIVT10, and plasminogen K4. We determined additionally the K(d) value of 0.31 +/- 0.04 mM for the binding of epsilon-aminocaproic acid (EACA) to apo(a) KIVT6 and mapped the binding region on apo(a) KIVT6 by means of chemical shift perturbation. This lysine binding activity, which was reported to occur within apo(a) KIVT5-8, is functionally different from the lysine binding activity found for apo(a) KIVT10.     

High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).     

Comparative analyses of the lysine binding site properties of apolipoprotein(a) kringle IV types 7 and 10.

Apolipoprotein(a) [apo(a)] shares extensive sequence similarity with plasminogen and consists of multiple tandem repeats of domains similar to plasminogen kringle IV (KIV), followed by domains homologous to the plasminogen KV and protease domains. The apo(a) KIV domains can be classified into 10 types on the basis of amino acid sequence (KIV(1)-KIV(10)) of which KIV(10) contains a canonical lysine binding site (LBS); KIV(10) mediates the lysine-dependent interaction of Lp(a) with certain biological substrates. Molecular modeling studies indicated the presence of weak LBS in each of KIV(5)-KIV(8), and subsequent biochemical studies have revealed contributions of these kringles to lysine-mediated interactions involving apo(a). The present study describes the direct demonstration of a weak LBS within KIV(7), as well as the first characterization of the ligand specificity of an LBS outside that of KIV(10). We have expressed both KIV(7) and KIV(10) from bacterial cells and purified them to homogeneity from cell lysates. Equilibrium binding analyses of the KIV(7) LBS using intrinsic fluorescence revealed an affinity for L-lysine and its analogues approximately 10-fold weaker (K(D) = 230 +/- 42 microM for epsilon-aminocaproic acid) than that of KIV(10) (K(D) = 33 +/- 4 microM for epsilon-aminocaproic acid). Moreover, we demonstrated differences in specificity of the LBS of KIV(7) in comparison with KIV(10) in that KIV(7) preferentially bound L-proline. Both kringles bind 4-aminobutyric acid with similar affinities albeit with apparently different mechanisms. Key Phe(62) --> Tyr and Asp(56) --> Glu substitutions in the KIV(7) LBS result in alterations in the size of the LBS and in the spatial relationship between the cationic and anionic centers in the LBS and thus account for the differences in the binding properties of KIV(7) and KIV(10).     

Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.     

Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance

The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.     

NMR solution structure and dynamics of an exchangeable apolipoprotein,Locusta migratoria apolipophorin III

We report here the NMR structure and backbone dynamics of an exchangeable apolipoprotein, apoLp-III, from the insect Locusta migratoria. The NMR structure adopts an up-and-down elongated five-helix bundle, which is similar to the x-ray crystal structure of this protein. A short helix, helix 4', is observed that is perpendicular to the bundle and fully solvent-exposed. NMR experimental parameters confirm the existence of this short helix, which is proposed to serve as a recognition helix for apoLp-III binding to lipoprotein surfaces. The L. migratoria apoLp-III helix bundle displays several characteristic structural features that regulate the reversible lipoprotein binding activity of apoLp-III. The buried hydrophilic residues and exposed hydrophobic residues readily adjust the marginal stability of apoLp-III, facilitating the helix bundle opening. Specifically, upon lipoprotein binding the locations and orientations of the buried hydrophilic residues modulate the apoLp-III helix bundle to adopt a possible opening at the hinge that is opposite the recognition short helix, helix 4'. The backbone dynamics provide additional support to the recognition role of helix 4' and this preferred conformational adaptation of apoLp-III upon lipid binding. In this case, the lipid-bound open conformation contains two lobes linked by hinge loops. One lobe contains helices 2 and 3, and the other lobe contains helices 1, 4, and 5. This preferred bundle opening is different from the original proposal on the basis of the x-ray crystal structure of this protein (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), but it efficiently uses helix 4' as the recognition short helix. The buried interhelical H-bonds are found to be mainly located between the two lobes, potentially providing a specific driving force for the helix bundle recovery of apoLp-III from the lipid-bound open conformation. Finally, we compare the NMR structures of Manduca sexta apoLp-III and L. migratoria apoLp-III and present a united scheme for the structural basis of the reversible lipoprotein binding activity of apoLp-III.     

NMR structure and dynamics of a receptor-active apolipoprotein E peptide

Apolipoprotein E (apoE) is important in lipid metabolism due to its interaction with members of the low density lipoprotein (LDL) receptor family. ApoE is able to interact with the LDL receptor only when it is bound to lipid particles. To address structural aspects of this phenomenon, a receptor-active apoE peptide, encompassing the receptor-binding region of the protein, was studied by NMR in the presence of the lipid-mimicking agent trifluoroethanol. In 50% trifluoroethanol, apoE-(126-183) forms a continuous amphipathic alpha-helix over residues Thr(130)-Glu(179). Detailed NMR relaxation analysis indicates a high degree of plasticity for the residues surrounding 149-159. This intrinsic flexibility imposes a curvature to the peptide that may be important in terms of interaction of apoE with various sized lipid particles and the LDL receptor. Residues 165-179 of apoE may act as a molecular switch whereby these residues are unstructured in the absence of lipids and prevent interaction with the LDL receptor. In the presence of lipids, these residues become helical resulting in a receptor-active conformation of the protein. Furthermore, the electrostatic characteristics and geometric features of apoE-(126-183) suggest that apoE binds to the LDL receptor by interacting with more than one of the receptor ligand-binding repeats.     

Quantitative evaluation of the contribution of weak lysine-binding sites present within apolipoprotein(a) kringle IV types 6-8 to lipoprotein(a) assembly

During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV(6-8)), and two lysine residues (Lys(680) and Lys(690)) within the NH(2) terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV(6-8) and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV(6), KIV(7), and KIV(8), as well as mutants that disrupt the WLBS in both KIV(6) and KIV(7), or both KIV(7) and KIV(8), were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV(7) and KIV(8), but not KIV(6), are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV(7) or KIV(8) resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV(7) and KIV(8) resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV(7) and KIV(8) specifically bind with high affinity to apoB-derived peptides containing Lys(690) or Lys(680), respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV(7) and KIV(8) and Lys(680) and Lys(690) in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.     

Nuclear magnetic resonance studies and molecular dynamics simulations of the solution conformation of a 'designed', alpha-helical peptide.

The complete three-dimensional structure in methanol of an amphipathic alpha-helical peptide, that has been designed by taking into account the three-dimensional structures of small haemolytic peptides, secondary structure prediction algorithms and the well documented literature on alpha-helix stabilizing factors, has been elucidated by two-dimensional NMR spectroscopy. Initially various two-dimensional spectra (COSY, TOCSY, and NOESY) allowed the complete sequence specific assignment of all signals in the 1H spectrum. Consequently trial structures were generated which were then subjected to molecular dynamics simulations using 121 NOE-derived distances and 25 vicinal coupling constant values as structural restraints to give a final set of calculated structures. These structures are in complete agreement with the results of a circular dichroism study and reveal that the peptide adopted a highly ordered alpha-helical conformation. Details of the structure which throw light on future peptide/protein design are discussed.     

Nuclear magnetic resonance (NMR) studies of the dark-color-inducing neurohormone of locusts and corazonin

The dark-color-inducing neurohormone (DCIN) of locusts and corazonin of a cockroach, both 11 residue-long peptides, induce dark coloration in albino nymphs of Locusta migratoria when injected after a nymphal molt. These peptides differ at position 7 (His in DCIN and Arg in corazonin) and elicit an almost identical darkening response. The three-dimensional structures of these peptides, dissolved in dimethylsulfoxide (DMSO), were determined by NMR. Structural elements determined at atomic resolution may provide insight into the biological activity of these two neurohormones. The calculated structures of DCIN and corazonin indicate clear, prevalent conformations with similar secondary features. The generated low-energy solution structures of each show structural elements within residues Phe3 to Trp9 with a turn situated at the core of the peptide from which the sidechains of residue 7 of each peptide protrude. A calculated negative electrostatic potential surface almost completely covers both neurohormones and only the 7th residue sidechains of each peptide emerge in their entirety. Within these residues there is a partial sequence seen in several neurohormones that control various physiological functions in Arthropods: -Ser-X-Gly-Trp- (X=His in DCIN and Arg in corazonin). This partial sequence may play a role in the physiological activity of some Arthropod neurohormones.     

Baboon lipoprotein(a) binds very weakly to lysine-agarose and fibrin despite the presence of a strong lysine-binding site in apolipoprotein(a) kringle IV type 10

Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. To further the comparative analyses with other primate species, we sequenced a segment of baboon liver apo(a) cDNA spanning KIV(9) through the protease domain. Like rhesus monkey apo(a), baboon apo(a) lacks a kringle V (KV)-like domain. Interestingly, we found that the baboon apo(a) KIV(10) sequence contains all of the canonical LBS residues. We sequenced the apo(a) KIV(10) sequence from an additional 10 unrelated baboons; 17 of 20 alleles encoded Trp at position 70, whereas only two alleles encoded Arg at this position and thus a defective LBS. Despite the apparent presence of a functional KIV(10) LBS in most of the baboons, none of the Lp(a) in the plasma of the corresponding baboons bound specifically to lysine-Sepharose (agarose) even upon partial purification. Moreover, baboon Lp(a) bound very poorly to plasmin-modified fibrinogen. Expression of baboon and human KIV(10) in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that presentation of KIV(10) in the context of apo(a) lacking KV may interfere with the ability of KIV(10) to bind to substrates such as fibrin; this paradigm may also be present in other non-human primates.     

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V)

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.     

Nuclear magnetic resonance study of the solution structure of alpha 1-purothionin. Sequential resonance assignment, secondary structure and low resolution tertiary structure

The solution structure of the 45-residue plant protein, alpha 1-purothionin, is investigated by nuclear magnetic resonance (n.m.r.) spectroscopy. Using a combination of two-dimensional n.m.r. techniques to demonstrate through-bond and through-space (less than 5 A) connectivities, the 1H n.m.r. spectrum of alpha 1-purothionin is assigned in a sequential manner. The secondary structure elements are then delineated on the basis of a qualitative interpretation of short-range nuclear Overhauser effects (NOE) involving the NH, C alpha H and C beta H protons. There are two helices extending from residues 10 to 19 and 23 to 28, two short beta-strands from residues 3 to 5 and 31 to 34 which form a mini anti-parallel beta-sheet, and five turns. In addition, a number of long-range NOE connectivities are assigned and a low resolution tertiary structure is proposed.     

Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.     

Molecular structure and physical properties of type IV collagen in solution

The physical properties of intact type IV collagen from the mouse EHS sarcoma were studied in acid solution using laser light scattering and viscometry. The experimentally observed values of molecular weight, translational diffusion coefficient, particle scattering factor at 175.5° and a wavelength of 633 nm and intrinsic viscosity at 22°C were 532000, 0.66 × 10⁻⁷cm²s⁻¹, 0.492 and 74.7 ml/g respectively. Plots of $\text{Kc/}\text{R}{}_0$ versus collagen concentration were linear with a slope of approximately 0, indicating that under the conditions studied, type IV collagen molecules do not form supra-molecular aggregates. Experimentally determined translational diffusion coefficients closely approximated the calculated value for a rod-like molecule 424 nm long and 1.5 nm in diameter. Based on this observation, it is concluded that the type IV collagen molecule translates like a bent rigid rod similar to the interstitial collagens. However, the low intrinsic viscosity and larger value of the particle scattering factor for type IV collagen molecules in comparison with the interstitial collagens indicate that type IV collagen is considerably more flexible. Physical measurements on molecules in solution are consistent with a model of the type IV molecule containing numerous flexible bends with bend angles less than 125°. It is concluded that the type IV collagen molecule behaves like a worm-like rod in solution.     

Nuclear magnetic resonance solution structure of PisI, a group B immunity protein that provides protection against the type IIa bacteriocin piscicolin 126, PisA

Lactic acid bacteria produce and secrete bacteriocins. These bacteriocins are potent antimicrobial peptides that are active against other closely related bacteria. As a means of self-protection, producer organisms also express immunity proteins. Immunity proteins are generally located on the same genetic locus and are cotranscribed with the bacteriocin. Although some cross immunity between bacteriocins has been observed, immunity proteins are typically highly specific. Immunity proteins for the type IIa bacteriocins range from 81 to 115 amino acids in length and display substantial variation in their sequences. Nonetheless, such immunity proteins have been classified into three groupings (groups A, B, and C) according to sequence homology. The structures of a group C (ImB2) and two group A (EntA-im and PedB) immunity proteins have previously been reported. We herein report the nuclear magnetic resonance solution structure of the remaining class of the type IIa immunity proteins. PisI, a 98-amino acid protein, is a group B immunity protein conferring immunity against piscicolin 126 (PisA). Like ImB2, EntA-im, and PedB, PisI folds into a globular protein in aqueous solution and contains an antiparallel four-helix bundle. Compared to ImB2 and EntA-im, PisI has a substantially longer and more flexible N-terminus, but a shorter C-terminus. No direct interaction between the bacteriocin and immunity protein is observed by NMR in either aqueous or membrane mimicking environments. This further suggests that the mechanism that mediates immunity is not due to a direct bacteriocin-immunity protein interaction but rather is receptor-mediated. It has now been confirmed that the four-helix bundle is indeed a structural motif among the type IIa immunity proteins.