Structural insights into molecular-targeting helix-loop-helix peptide against vascular endothelial growth factor-A.

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5.

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Crystallization of Human Erythrocyte Band 3, the anion exchanger, at the International Space Station "KIBO".

Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.

Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and thermolysin.

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blue-shifted with increasing concentrations (0-2.0 µM) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (Kd) of the complex between ANS and thermolysin was 33 ± 2 µM at 0 M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9 ± 3 µM at 4 M NaCl. The Kd values were not varied (31-34 µM) in a pH range of 5.5-8.5. This suggests that at high NaCl concentrations, Na(+) and/or Cl(-) ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na(+) and/or Cl(-) ions with thermolysin.

Novel protein fold discovered in the PabI family of restriction enzymes.

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction-modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel beta-sheet that forms a 'half pipe'. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg(2+). These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.

Crystal structures of the short-chain flavin reductase HpaC from Sulfolobus tokodaii strain 7 in its three states: NAD(P)(+)(-)free, NAD(+)(-)bound, and NADP(+)(-)bound.

4-Hydroxyphenylacetate (4-HPA) is oxidized as an energy source by two component enzymes, the large component (HpaB) and the small component (HpaC). HpaB is a 4-HPA monooxygenase that utilizes FADH(2) supplied by a flavin reductase HpaC. We determined the crystal structure of HpaC (ST0723) from the aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 in its three states [NAD(P)(+)-free, NAD(+)-bound, and NADP(+)-bound]. HpaC exists as a homodimer, and each monomer was found to contain an FMN. HpaC preferred FMN to FAD because there was not enough space to accommodate the AMP moiety of FAD in its flavin-binding site. The most striking difference between the NAD(P)(+)-free and the NAD(+)/NADP(+)-bound structures was observed in the N-terminal helix. The N-terminal helices in the NAD(+)/NADP(+)-bound structures rotated ca. 20 degrees relative to the NAD(P)(+)-free structure. The bound NAD(+) has a compact folded conformation with nearly parallel stacking rings of nicotinamide and adenine. The nicotinamide of NAD(+) stacked the isoalloxazine ring of FMN so that NADH could directly transfer hydride. The bound NADP(+) also had a compact conformation but was bound in a reverse direction, which was not suitable for hydride transfer.

Preliminary X-ray crystallographic analysis of thermolysin in the presence of 4 M NaCl.

The activity of thermolysin (EC 3.4.24.27) is greatly enhanced by high concentrations of neutral salts. For instance, 4 M NaCl enhances the activity 13-15-fold [Holmquist & Vallee (1976), Biochemistry, 15, 101-107; Inouye (1992), J. Biochem. (Tokyo), 112, 335-340]. To clarify the structural basis of the activation of thermolysin by high concentrations of NaCl, we have developed a new method to introduce 4 M NaCl into the P6(1)22 crystal of thermolysin originally grown without NaCl. The crystal obtained by this method diffracted X-rays to 2.43 A. No unit-cell parameter change was observed except the length of the c axis, which was elongated by 9.6% by the introduction of 4 M NaCl.

Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Thermus thermophilus HB8.

Peptide deformylase (PDF) is responsible for cleaving the formyl group at the N-terminus of nascent polypeptide chains in eubacteria and is essential to bacterial cell viability. A recombinant PDF of the thermophilic bacterium Thermus thermophilus HB8 has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belonged to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 62.58, c = 105.27 A, and are most likely to contain two molecules in an asymmetric unit, giving a crystal volume per protein weight (V(M)) of 2.3 A(3) Da(-1) and a solvent content of 46.7%.

Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus.

Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals diffracted X-rays to beyond 2.6 A resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 A. The crystal contains three molecules in an asymmetric unit (VM = 2.11 A3 Da(-1)) and has a solvent content of 61.5%.