Peter J. Steinbach

New spherical-cutoff methods for long-range forces in macromolecular simulation

New atom‐ and group‐based spherical‐cutoff methods have been developed for the treatment of nonbonded interactions in molecular dynamics (MD) simulation. A new atom‐based method, force switching, leaves short‐range forces unaltered by adding a constant to the potential energy, switching forces smoothly to zero over a specified range. A simple improvement to group‐based cutoffs is presented: Switched group‐shifting shifts the group–group potential energy by a constant before being switched smoothly to zero. Also introduced are generalizations of atom‐based force shifting, which adds a constant to the Coulomb force between two charges. These new approaches are compared to existing methods by evaluating the energy of a model hydrogen‐bonding system consisting of two N‐methyl acetamide molecules and by full MD simulation. Thirty‐five 150 ps simulations of carboxymyoglobin (MbCO) hydrated by 350 water molecules indicate that the new methods and atom‐based shifting are each able to approximate no‐cutoff results when a cutoff at or beyond 12 Å is used. However, atom‐based potential‐energy switching and truncation unacceptably contaminate group–group electrostatic interactions. Group‐based potential truncation should not be used in the presence of explicit water or other mobile electrostatic dipoles because energy is not a state function with this method, resulting in severe heating (about 4 K/ps in the simulations of hydrated MbCO). The distance‐dependent dielectric (ϵ ∝︁ r) is found to alter the temperature dependence of protein dynamics, suppressing anharmonic motion at high temperatures. Force switching and force shifting are the best atom‐based spherical cutoffs, whereas switched group‐shifting is the preferred group‐based method. To achieve realistic simulations, increasing the cutoff distance from 7.5 to 12 Å or beyond is much more important than the differences among the three best cutoff methods.

Identification of the Cysteine Residues in the Amino-terminal Extracellular Domain of the Human Ca2+ Receptor Critical for Dimerization IMPLICATIONS FOR FUNCTION OF MONOMERIC Ca2+RECEPTOR

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca2+ receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca2+ response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a “venus flytrap” model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca2+ response of the receptor.

Solid–state NMR evidence for an antibody–dependent conformation of the V3 loop of HIV–1 gp120

Solid–state NMR measurements have been carried out on frozen solutions of the complex of a 24–residue peptide derived from the third variable (V3) loop of the HIV–1 envelope glycoprotein gp120 bound to the Fab fragment of an anti–gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody–bound state. In combination with earlier crystal structures of V3 peptide–antibody complexes and existing data on the cross–reactivity of the antibodies, the solid–state NMR measurements suggest that the Gly–Pro–Gly–Arg (GPGR) motif adopts an antibody–dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full–length gp120. These measurements are the first application of solid–state NMR methods in a structural study of a peptide–protein complex.

The effects of environment and hydration on protein dynamics: A simulation study of myoglobin

Abstract Three classes of molecular dynamics (MD) simulations of carboxy-myoglobin (MbCO) have been performed to investigate the environmental and temperature dependence of protein dynamics. The first class examines the effects of hydration. Simulations of MbCO were performed at 100 and 300 K with 0, 35, 100, 349, and 999 water molecules, and at 300 K with 3832 water molecules. The second class considers a cluster of three partially hydrated MbCO molecules (349 waters each) at 100, 180, 240, and 300 K. The third class of simulations, performed at 100 and 300 K, examines hydration by D2O and also the effects of different vacuum models and long-range electrostatic cutoff methods. The simulations generally consist of 200 ps of heating and equilibration followed by 100 ps of dynamics used for analysis. Atomic fluctuation is compared to neutron scattering data to better determine the type of calculation needed to reproduce the low-temperature behavior of proteins. The simulations of hydrated myoglobin indicate that as the hydration increases: (i) agreement with the X-ray structure improves, (ii) the number of heavy-atom dihedral transitions decreases at both 100 and 300 K, and (iii) atomic fluctuations decrease at 100 K but increase at 300 K. The cluster and deuterated simulations exhibit atomic fluctuations at 100 K similar to, but slightly reduced from, those of a single hydrated myoglobin. Thus, our previous observation from MD simulations of low-temperature mean-square fluctuation three times larger than that observed experimentally does not appear to be due to the mass of the water model, nor the complete absence of intermolecular protein-protein contacts.

Transglutaminase-1 gene mutations in autosomal recessive congenital ichthyosis: summary of mutations (including 23 novel) and modeling of TGase-1.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification diseases. Germline mutations in TGM1 are the most common cause of ARCI in the United States. TGM1 encodes for the TGase‐1 enzyme that functions in the formation of the cornified cell envelope. Structurally defective or attenuated cornified cell envelop have been shown in epidermal scales and appendages of ARCI patients with TGM1 mutations. We review the clinical manifestations as well as the molecular genetics of ARCI. In addition, we characterized 115 TGM1 mutations reported in 234 patients from diverse racial and ethnic backgrounds (Caucasion Americans, Norwegians, Swedish, Finnish, German, Swiss, French, Italian, Dutch, Portuguese, Hispanics, Iranian, Tunisian, Moroccan, Egyptian, Afghani, Hungarian, African Americans, Korean, Japanese and South African). We report 23 novel mutations: 71 (62%) missense; 20 (17%) nonsense; 9 (8%) deletion; 8 (7%) splice‐site, and 7 (6%) insertion. The c.877‐2A>G was the most commonly reported TGM1 mutation accounting for 34% (147 of 435) of all TGM1 mutant alleles reported to date. It had been shown that this mutation is common among North American and Norwegian patients due to a founder effect. Thirty‐one percent (36 of 115) of all mutations and 41% (29 of 71) of missense mutations occurred in arginine residues in TGase‐1. Forty‐nine percent (35 of 71) of missense mutations were within CpG dinucleotides, and 74% (26/35) of these mutations were C>T or G>A transitions. We constructed a model of human TGase‐1 and showed that all mutated arginines that reside in the two beta‐barrel domains and two (R142 and R143) in the beta‐sandwich are located at domain interfaces. In conclusion, this study expands the TGM1 mutation spectrum and summarizes the current knowledge of TGM1 mutations. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of 5′ methylated CpG dinucleotides. Hum Mutat 0, 1–12, 2009.

The Tensin-3 Protein, Including its SH2 Domain, Is Phosphorylated by Src and Contributes to Tumorigenesis and Metastasis

In cell lines from advanced lung cancer, breast cancer, and melanoma, endogenous tensin-3 contributes to cell migration, anchorage-independent growth, and tumorigenesis. Although SH2 domains have not been reported previously to be phosphorylated, the tensin-3 SH2 domain is a physiologic substrate for Src. Tyrosines in the SH2 domain contribute to the biological activity of tensin-3, and phosphorylation of these tyrosines can regulate ligand binding. In a mouse breast cancer model, tensin-3 tyrosines are phosphorylated in a Src-associated manner in primary tumors, and experimental metastases induced by tumor-derived cell lines depend on endogenous tensin-3. Thus, tensin-3 is implicated as an oncoprotein regulated by Src and possessing an SH2 domain with a previously undescribed mechanism for the regulation of ligand binding.

Identification of Substrate Binding Site of Cyclin-dependent Kinase 5

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis (“charged to alanine”) and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.

Ω-crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin.

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Ω-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Ω-crystallin (officially designated ALDH1A9) is 55–56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/η-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Ω-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Ω-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Ω-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Ω-crystallins, which are tetrameric proteins, scallop Ω-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Redesign of a Four-Helix Bundle Protein by Phage Display Coupled with Proteolysis and Structural Characterization by NMR and X-ray Crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the proteins physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the proteins global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA–DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA–DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.