Kunihiro Kitamura

Charge Pairing of Headgroups in Phosphatidylcholine Membranes: A Molecular Dynamics Simulation Study

Molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase was carried out for 5 ns to study the interaction among DMPC headgroups in the membrane/water interface region. The phosphatidylcholine headgroup contains a positively charged choline group and negatively charged phosphate and carbonyl groups, although it is a neutral molecule as a whole. Our previous study (Pasenkiewicz-Gierula, M., Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi. 1997. J. Phys. Chem. 101:3677-3691) showed the formation of water cross-bridges between negatively charged groups in which a water molecule is simultaneously hydrogen bonded to two DMPC molecules. Water bridges link 76% of DMPC molecules in the membrane. In the present study we show that relatively stable charge associations (charge pairs) are formed between the positively and negatively charged groups of two DMPC molecules. Charge pairs link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together form an extended network of interactions among DMPC headgroups linking 98% of all membrane phospholipids. The average lifetimes of DMPC-DMPC associations via charge pairs, water bridges and both, are at least 730, 1400, and over 1500 ps, respectively. However, these associations are dynamic states and they break and re-form several times during their lifetime.

DEVELOPMENT OF MD ENGINE : HIGH-SPEED ACCELERATOR WITH PARALLEL PROCESSOR DESIGN FOR MOLECULAR DYNAMICS SIMULATIONS

Application of molecular dynamics (MD) simulations to large systems, such as biological macromolecules, is severely limited by the availability of computer resources. As the size of the system increases, the number of nonbonded forces (Coulombic and van der Waals interactions) to be evaluated increases as 𝒪(N2), where N is the number of particles in the system. The force evaluation consumes more than 99% of the CPU time in an MD simulation involving over 10,000 particles. Hence, the major target for reduction of the CPU time should be acceleration of the calculation of nonbonded forces. For this purpose, we developed a custom processor for calculating nonbonded interactions and a scalable plug‐in machine (to a workstation), the MD Engine, in which numbers of the custom processors work in parallel. The processor has a pipeline architecture to calculate the total nonbonded force using the coordinates, electric charge, and species of each particle broadcast by the host computer. The force is calculated with sufficient accuracy for practical MD simulations. The processor also calculates virials simultaneously with forces for use in the calculation of pressure, accommodates periodic boundary conditions, and can be used in Ewald summations. An MD Engine system consisting of 76 processors calculates nonbonded interactions about 50 times faster than an UltraSPARC‐I processor (Sun Ultra‐2, 200 MHz) or an R10000 processor (SGI Origin 200, 180 MHz). On a Sun Ultra‐2 workstation with a single UltraSPARC‐I processor an MD simulation of a Ras p21 protein molecule immersed in a water sphere (13,258 particles) was accelerated by a factor of 48 using the MD Engine system. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 185–199, 1999

Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites.

In order to investigate the binding mode of E64-c (a synthetic cysteine proteinase inhibitor) to papain at the atomic level, the crystal structure of the complex was analysed by X-ray diffraction at 1.9 A (1 A is expressed in SI units as 0.1 nm) resolution. The crystal has a space group P2(1)2(1)2(1) with a = 43.37, b = 102.34 and c = 49.95 A. A total of 21,135 observed reflections were collected from the same crystal, and 14811 unique reflections of up to 1.9 A resolution [Fo > 3 sigma(Fo)] were used for the structure solution and refinement. The papain structure was determined by means of the molecular replacement method, and then the inhibitor was observed on a (2 magnitude of Fo-magnitude of Fc) difference Fourier map. The complex structure was finally refined to R = 19.4% including 207 solvent molecules. Although this complex crystal (Form II) was polymorphous as compared with the previously analysed one (Form I), the binding modes of leucine and isoamylamide moieties of E64-c were significantly different from each other. By the calculation of accessible surface area for each complex atom, these two different binding modes were both shown to be tight enough to prevent the access of solvent molecules to the papain active site. With respect to the E64-c-papain binding mode, molecular-dynamics simulations proposed two kinds of stationary states which were derived from the crystal structures of Forms I and II. One of these, which corresponds to the binding mode simulated from Form I, was essentially the same as that observed in the crystal structure, and the other was somewhat different from the crystal structure of Form II, especially with respect to the binding of the isoamylamide moiety with the papain S subsites. The substrate specificity for the papain active site is discussed on the basis of the present results.

Molecular Dynamics Generation of Nonarbitrary Membrane Models Reveals Lipid Orientational Correlations

This report addresses the following problems associated with the generation of computer models of phospholipid bilayer membranes using molecular dynamics simulations: arbitrary initial structures and short equilibration periods, an Ewald-induced strong coupling of phospholipids, uncertainty regarding which value should be used for surface tension to alleviate the problem of the small size of the membrane, and simultaneous realization of both order parameters and the surface area. We generated a computer model of the liquid-crystalline L-alpha-dimyristoylphosphatidylcholine (DMPC) bilayer, starting from a configuration based on a crystal structure (rather than from an arbitrary structure). To break the crystalline structure, a 20-ps high-temperature pulse of 510 K (but not 450 or 480 K) was effective. The system finally obtained is an all-atom model, with Ewald summation to evaluate Coulombic interactions and a constant surface tension of 35 dynes/cm/water-membrane interface, equilibrated for 12 ns (over 50 ns total calculation time), which reproduces all of the experimentally observed parameters examined in this work. Furthermore, this model shows the presence of significant orientational correlations between neighboring alkyl chains and between shoulder vectors (which show the orientations of the lipids about their long axes) of neighboring DMPCs.

Antibiotic effect of linolenic acid fromChlorococcum strain HS-101 andDunaliella primolecta on methicillin-resistantStaphylococcus aureus

Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains wasα-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of γ-linolenic acid.

Mode of binding of E-64-c, a potent thiol protease inhibitor, to papain as determined by X-ray crystal analysis of the complex

The three‐dimensional structure of the E‐64‐c‐papain complex has been determined by X‐ray crystal analysis at 2.5 Å resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E‐64‐c is covalently bound by the Sγ atom of Cys‐25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S‐ to the R‐form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E‐64‐c molecule with papain.

Fast Lipid Disorientation at the Onset of Membrane Fusion Revealed by Molecular Dynamics Simulations

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.

How does the Electrostatic Force Cut-Off Generate Non-uniform Temperature Distributions in Proteins?

Abstract Molecular dynamics simulations (MDS) with electrostatic force cut-off on heterogeneous molecular systems make temperature separation between solute and solvent [1 –4]. In MDS where the temperature separation occurs, we found that there is a non-uniform temperature distribution in protein. The pattern of temperature distribution was shown to be peculiar to the protein, suggesting a possible relationship between dynamical and thermal properties of protein. By principal component analysis, the non-uniform temperature distribution was ascribed to high temperature of large fluctuation modes in protein. Further analysis by thermal diffusion equation between principal modes showed that in protein large fluctuation modes inherently have smaller value of heat capacity than small fluctuation modes have and are easily heated up by truncation noise due to abrupt cut-off of electrostatic forces.

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed. Murine Uld consists of two alpha helices [Ile23-Arg33 (alpha1) and Val56-Gln57 (alpha2)] and five beta strands [Met1-Phe7 (beta1), Tyr11-Asp18 (beta2), Leu41-Phe45 (beta3), Lys48-Pro51 (beta4) and Ser65-Arg72 (beta5)] and its overall structure is essentially the same as that of human ubiquitin with a 1.22 A rmsd for the backbone atoms of residues 1-76; however, the sequential identity and similarity between both molecules are 32% and 63%, respectively. This close resemblance is due to the core structure built by same hydrogen bond formations between and within the backbone chains of alpha1 and beta1/2/5 secondary structure elements and by nearly the same hydrophobic interactions formed between the nonpolar amino acids of their secondary structures. The side chain NetaH of Lys27 on the alpha1 helix was crucial to the stabilization of the spatial orientations of beta3 and beta4 strands, possible binding region with Rpn 10 subunit, through three hydrogen bonds. The MD simulations showed the K27N and R33Q mutations increase the structural fluctuation of these beta strands including the alpha1 helix. Reversely, the V56E mutant restricted the spatial flexibility at the periphery of the short alpha2 helix by the interactions between the polar atoms of Glu56 and Ser19 residues. However, a large fluctuation of beta4 strand with respect to beta5 strand was induced in the R42P mutant, because of the impossibility of forming paired hydrogen bonds of Pro for Arg42 in wild Uld. The X-ray structure showed that the side chains of Asp39, Gln40 and Arg42 at the N-terminal periphery of beta3 strand protrude from the molecular surface of Uld and participate in hydrogen bonds with the polar residues of neighboring Ulds. Thus, the MD simulation suggests that the mutation substitution of Pro for Arg42 not only causes the large fluctuation of beta3 strand in the Uld but also leads to the loss of the ability of Uld to trap the Rpn 10 subunit. In contrast, the MD simulation of K48A mutant showed little influence on the beta3-beta4 loop structure, but a large fluctuation of Lys48 side chain, suggesting the importance of flexibility of this side chain for the interaction with the Rpn 10 subunit. The present results would be important in elucidating the impaired proteasomal binding mechanism of parkin in AR-JP.

Error evaluation in the design of a special-purpose processor that calculates nonbonded forces in molecular dynamics simulations

Special‐purpose parallel machines that are plugged into a workstation to accelerate molecular dynamics (MD) simulations are attracting a considerable amount of interest. These machines comprise scalable homogeneous multiprocessors for calculating nonbonded forces (Coulombic and van der Waals forces), which consume more than 99% of the central processing unit (CPU) time in standard MD simulations. Each processor element in the machine has a pipeline architecture to calculate the total nonbonded force exerted on a particle by all of the other particles using information regarding the coordinates, the electric charge, and the species of each particle broadcast by the host computer. The processor then sends the calculated force back to the host computer. This article addresses the precision of the calculated nonbonded forces in the design of a processor LSI with minimal complexity. The precision of the arithmetic inside the processor that is required to calculate forces for MD simulations using Verlets procedure was critically evaluated. Forward and backward error analysis, coupled with numerical MD experiments on one‐dimensional systems, was performed, and the following results were obtained: (1) Each element of the position vector which the processor receives from the host computer should have a precision of at least 25 bits; and (2) the pairwise forces should be calculated using floating point numbers with at least 29 bits of mantissa in the processor. Calculation of a pairwise force, which involves second‐order polynomial interpolation using a table‐driven algorithm, requires a key which contains a duplicate of at least 11 most significant bits of mantissa of the squared pairwise distance. The final result was that (3) the total force that acts on a particle, which is obtained by summing the forces exerted by all of the other particles, should be calculated using an accumulator that has a mantissa of at least 48 bits.