Yasuhiro Kumaki

Flexible Structures and Ligand Interactions of Tandem Repeats Consisting of Proline, Glycine, Asparagine, Serine, and/or Threonine Rich Oligopeptides in Proteins

Tandem repeats occur in 14% of all proteins. The repeat unit lengths range from a single amino acid to more than 100 residues and the repeat number is sometimes over 100. Understanding the structures, functions, and evolution of these repeats is a significant goal in both proteomics and genomics. This review summarizes experimental studies addressing structural features of tandem repeats of short oligopeptides that are rich in proline, glycine, asparagine, serine, and/or threonine. The oligopetides include (PGMG) and (PNN) in biomineralization protein (PM27), and (NPNA) in Plasmodium falciparum circumsporozoite protein, (YSPTSPS) in RNA polymerase II, (PHGGGWGQ) in the prion protein, (YGHGGG(N)) and (YNHGGG(G)) in plant glycine-rich proteins, (PGQGQQ), (PGQGQQGQQ) and (GYYPTSOQQ) of wheat HMW glutenin, (FGGMGGGKGG) in Aequipecten abductin. Spectroscopic studies including NMR and CD indicate that these peptides adopt type I and II beta-turns, polyproline II helices, loop conformations, and random coils. Formation of these structures frequently depends on pH, solvent, temperature and hydration. The loop conformations are sometimes stabilized by cation-phi, CH-phi, and/or amino-aromatic interactions. These observations indicate that many tandem repeats are largely flexible. In addition to generating repeating domains and providing flexible linkers between domains, the tandem repeats of (PHGGGWGQ), (YGHGGG(N)) and (YNHGGG(G)) and those in titin bind Cu(2+) ions; whereas, tandem repeats of (NPNA) and those in elastin bind Ca(2+) ions. The interactions of some tandem repeats with various target proteins probably involve an induced fit. The tandem repeats in tropoelastin, flagelliform silk, wheat HMW glutenin, abductin, titin, and human nucleoporin, nup153, are responsible for elastomeric properties.

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded β-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist ω-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that ω-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and ω-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and ω-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and ω-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe9 of tachystatin A might be an essential residue for binding to chitin.

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.

Hydrogen exchange study of canine milk lysozyme: Stabilization mechanism of the molten globule

The native state 1H, 15N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and α‐lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two‐state model, whereas that of α‐lactalbumin follows a three‐state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of α‐lactalbumin but with extreme stability. Study of the molten globules of α‐lactalbumin and equine milk lysozyme showed that the stabilities of their α‐helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the α‐helices are more stabilized than in α‐lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state. Proteins 2000;40:579–589.

X-ray crystallography and structural stability of digestive lysozyme from cow stomach.

In ruminants, some leaf‐eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.

A circular loop of the 16-residue repeating unit in ice nucleation protein.

Ice nucleation protein (INP) from Gram-negative bacteria promotes the freezing of supercooled water. The central domain of INPs with 1034-1567 residues consists of 58-81 tandem repeats with the 16-residue consensus sequence of AxxxSxLTAGYGSTxT. This highly repetitive domain can also be represented by tandem repeats of 8-residues or 48-residues. In order to elucidate the structure of the tandem repeats, NMR measurements were made for three synthetic peptides including QTARKGSDLTTGYGSTS corresponding to a section of the repetitive domains in Xanthomonas campestris INP. One remarkable observation is a long-range NOE between the side chains of Tyr(i) and Ala(i-10) in the 17-residue peptide. Medium-range NOEs between the side chains of Tyr(i) and Leu(i-4), Thr(i-3) or Thr(i-2) were also observed. These side chain-side chain interactions can be ascribed to CH/pi interaction. Structure calculation reveals that the 17-residue peptide forms a circular loop incorporating the 11-residue segment ARKGSDLTTGY.

Design and Synthesis of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose, a Calcium Ion-Mobilizing Second Messenger†

Cyclic ADP-ribose (cADPR, 1, Scheme 1), originally isolated from sea urchins by Lee and co-workers,1 is a general mediator of intracellular Ca2+ ion signaling.2 Analogues of cADPR have been extensively designed and synthesized3, 4 because of their potential usefulness for investigating the mechanisms of cADPR-mediated Ca2+ release and application as lead structures for the development of drug candidates.2

Structural properties of the DNA-bound form of a novel tandem repeat DNA-binding domain, STPR.

Fibroin‐modulator‐binding protein 1 (FMBP‐1) is a predicted transcription factor of the silkworm fibroin gene. The DNA‐binding domain of FMBP‐1 consists of four almost perfect tandem repeats of 23 amino acids each (R1–R4), and is referred to as the score and three amino acid peptide repeat (STPR) domain. This characteristic domain is conserved in eukaryotes, but the DNA‐binding mode is not known. In this study, the structural properties of the DNA‐bound form of the STPR domain were characterized. The combined experiments indicated that the STPR domain bound to the DNA duplex with a 1:1 binding ratio. The specific DNA caused considerable changes in the thermal unfolding profile and the digestion pattern of the STPR domain. These data suggested that the domain adapts a quite rigid helix‐rich structure in the DNA‐bound state, even though it moves flexibly in the absence of DNA. Furthermore, mutual induced‐fit conformational change was also observed in DNA. Finally, we determined the DNA‐binding surface of the STPR third repeat (R3) by alanine scanning mutagenesis; a particular site, composed of hydrophobic and hydrophilic residues, was identified. Notably, the substitution of Arg‐9 in R3 with alanine residue, which is located in the middle of the surface, drastically abolished the α‐helix‐inducing and DNA‐binding abilities. From these results, we predicted the DNA‐binding mode of the STPR domain. Proteins 2008.

Effects of a helix substitution on the folding mechanism of bovine α‐lactalbumin

The structure, stability, and unfolding‐refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105–110) comes from equine lysozyme and the remainder of the sequence comes from bovine α‐lactalbumin were studied by circular dichroism spectroscopy and stopped‐flow measurements, and the results were compared with those of bovine α‐lactalbumin. The substitution of the flexible loop in bovine α‐lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine α‐lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine α‐lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non‐native interaction in the flexible loop region and that the disruption of the non‐native interaction reduces the free energy barrier of refolding. We conclude that the non‐native interaction in the molten globule state may act as a kinetic trap for the folding of α‐lactalbumin. Proteins 2002;49:95–103.

Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed 15N‐labeled and 13C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed 15N‐edited and 13C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright