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Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this ‘slip’ mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a ‘detoxification’ mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development., Author summary There is an important group of proteins, secondary active transporters, that are integral parts of membranes and that act as molecular pumps to move specific molecules, often referred to as cargo, across those membranes. This is an active process that typically exploits the energy inherent in the ionic gradients cells maintain across their membranes. Many transporters bind their cargos weakly because this is a physical requirement for rapid transport and this creates a problem that they would then fail to discriminate between the correct cargo and chemically similar molecules that would have a harmful effect if transported. Here, we explore the hypothesis that this discrimination can be enhanced by biochemical processes known in the literature as “proofreading” and/or “editing”. This exploration includes the expansion of an established kinetic model of a known transporter from V. parahaemolyticus (vSGLT) that transports sugars; mathematical analysis of the expanded model; numerical solution of the differential equations at the core of the expanded model; and a combination of data-base searches and molecular docking studies that establish the existence of toxic molecules sufficiently similar to the target cargo that they could be transported by vSGLT. We conclude that proofreading/editing is a plausible addition to the repertoire of secondary active transporters that would resolve the discrimination conundrum described above. We also note that the specifics of the model reported here reveal an interesting aspect of proofreading/editing behavior (unbounded selectivity) that, to our knowledge, has not been reported previously.

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a “competitive” environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest., Author summary Molecular machines, which operate on the nanoscale, are proteins/complexes that perform remarkable tasks such as the selective absorption of nutrients into the cell by transporters. These complex machines are often described using a fairly simple set of states and transitions that may not account for the stochasticity and heterogeneity generally expected at the nanoscale at body temperature. New tools are needed to study the full array of possibilities. This study presents a novel in silico method to systematically generate testable molecular-machine kinetic models and explore alternative mechanisms, applied first to membrane transport proteins. Our initial results suggest these transport machines may contain mechanisms which ‘detoxify’ the cell of an unwanted toxin, as well as significantly discriminate against the import of the toxin. This novel approach should aid the experimental study of key physiological processes such as renal glucose re-absorption, rational drug design, and potentially the development of synthetic machines.

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a “competitive” environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.Author summaryMolecular machines, which operate on the nanoscale, are proteins/complexes that perform remarkable tasks such as the selective absorption of nutrients into the cell by transporters. These complex machines are often described using a fairly simple set of states and transitions that may not account for the stochasticity and heterogeneity generally expected at the nanoscale at body temperature. New tools are needed to study the full array of possibilities. This study presents a novel in silico method to systematically generate testable molecular-machine kinetic models and explore alternative mechanisms, applied first to membrane transport proteins. Our initial results suggest these transport machines may contain mechanisms which ‘detoxify’ the cell of an unwanted toxin, as well as significantly discriminate against the import of the toxin. This novel approach should aid the experimental study of key physiological processes such as renal glucose re-absorption, rational drug design, and potentially the development of synthetic machines.

Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.

The energy surface in the vicinity of the "Eco RI kink" was investigated by conducting both in vacuo molecular dynamics simulations as well as a simulation with explicit solvent. The in vacuo simulations used the "all atom" AMBER 3.0 force field with a distant dependent dielectric function and "hydrated" counter ions while the simulation with explicit solvent used the AMBER 4.0 force field, fully charged phosphates and counter ions and a dielectric constant of 1.0. The thrust of the simulations was to discriminate between two models of the energy surface of the deformed DNA as found in the recognition complex with Eco RI endonuclease. In the intrinsic model, the kinked DNA is a local minimum of the energy surface intrinsic to the DNA itself while in the strained model there is no significant energy barrier separating kinked and regular B-DNA. The two models have significant implications for theories of indirect recognition of DNA based on sequence-dependent deformability. The simulations suggest that the Eco RI-kinked structure is an example of molecular strain because it is not near a minimum of any of the potential energy functions examined. The simulations leave the question of an energy barrier somewhat open and raise the possibility that the Eco RI kink is at (or near) a point of dynamic instability of the energy surface (either a true maximum or a saddle point).

The past year has seen significant advances in our understanding of the structure and function of restriction endonucleases. The highlights include a revised chain tracing for Eco RI endonuclease from Escherichia coli , structures soon to be reported for E. coli Eco RV endonuclease and significant advances in the biochemistry and molecular genetics of both enzymes.

The free energy of the binding reaction between EcoRI restriction endonuclease and a specific cognate dodecadeoxynucleotide (d(CGCGAATTCGCG)) has contributions from both electrostatic and nonelectrostatic components. These contributions were dissected by measuring the effects of varying salt concentration on the equilibrium binding constant and applying the thermodynamic analyses of Record et al. (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158). Endonuclease mutation S187 (Arg 187 to Ser) (Greene, P. J., Gupta, M., Boyer, H. W., Brown, W. E., and Rosenberg, J. M. (1981) J. Biol. Chem. 256, 2143-2153) did not significantly affect the nonelectrostatic component but did perturb the electrostatic contribution to the binding energy (we are numbering the amino acid residues according to the DNA sequence). The former was determined by extrapolating the linear portion of the salt dependence curve (0.125 to 0.25 M KCl) to 1 M ionic strength, with the same result for both wild type and S187 endonucleases at both pH 6.0 and 7.4 (-8.5 +/- 1.5 kcal/mol or greater than 50% of the total binding free energy). The slopes of these same curves yield estimates of eight ionic interactions between wild type endonuclease and the DNA at both pH values. By contrast, binding of EcoRI-S187 to dodecanucleotide involves six charge-charge interactions at pH 6.0. Only two ionic interactions are observed at pH 7.4. This was unexpected since gel permeation chromatography demonstrated that the recognition complex for both wild type and S187 proteins contains an enzyme dimer and a DNA duplex. EcoRI-S187 endonuclease retains wild type DNA sequence specificity, and the rate of the phosphodiester hydrolysis step is also unchanged. Thus, electrostatic interactions are functionally separable from sequence recognition and strand cleavage. Our results also establish that arginine 187 plays a key role in the electrostatic function and suggest that it might be located at the DNA-protein interface. The disproportionate loss of ion pairs at pH 7.4 can be rationalized by a model which suggests that six conformationally mobile ionic groups on the protein act in a coordinated manner during the interaction with DNA.

Optimum coordinate sets have been obtained for ferrocytochrome c and the two symmetry-independent molecules of ferricytochrome c from tuna at 2.0 A resolution by making the best fit of models with standard bond lengths and angles to the experimental electron density maps (1977) J. Biol. Chem. 252, 759-785, as a preliminary to full refinement with 1.5 A data. Both the Diamond model-building programs and locally developed minicomputer routines were tried, with the latter preferred for economy and ease of operation, although both gave satisfactory results. Atomic coordinates are available on microfiche or from the Brookhaven Protein Data Bank. Using the two ferricytochrome molecules as a control, no differences between oxidized and reduced cytochrome molecules can be seen that are outside the probable limits of accuracy of the 2.0 A analysis. Rotation and subtractive difference map comparisons also show no conformation changes. If believable differences do appear in the course of the 1.5 A refinement now underway, these should be no more than minor breathing of main chain or adjustment of side chains.

The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.

The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases the affinity of the protein for inducer molecules. The maximum change observed is a 20-fold increase in the concentration of inducer necessary for half-saturation of the protein. The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating amounts of operator DNA fragments were compared to curves predicted by several different models. Using the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to discriminate between the various models. The models which accurately predicted the inducer binding curves and conformed to the measured values for the other parameters were Monod-Wyman-Changeux and Koshland type models with the following features: 1) positive cooperativity in the binding of inducer to the repressor . operator DNA complex; 2) two sites for the binding of the operator DNA fragment to repressor protein; 3) effect of binding of each operator DNA fragment on all four subunits of the repressor. These models are consistent with all of the available data, and they indicate that the binding of the first two inducer molecules accounts for greater than or equal to 60% of the difference in affinities between free and induced repressor for operator DNA; in vivo this difference should be sufficient for induction to occur.

The rates of binding of inducer molecules to the lactose repressor protein are significantly affected by the presence of bound operator DNA fragments. The association rate is decreased from 4.6 x 10(4) M-1S-1 to 1.0 x 10(4) M-1S-1 by the presence of saturating amounts of operator DNA fragments. The inducer dissociation rate was measured by dilution of repressor . operator . inducer complexes and by displacement of the sugar molecule from repressor by the binding of operator DNA. The value for the dissociation rate in the presence of bound DNA was 0.8 S-1; this is 4-fold greater than the inducer dissociation rate measured in the absence of operator. These kinetic results suggest that repressor should exhibit a 20-fold lower affinity for inducer when operator DNA fragments are bound; this prediction is in agreement with previous equilibrium measurements. Measurement of the rate of operator fragment dissociation from repressor using a nitrocellulose filter assay yields a value of 0.04 S-1 in the absence of inducer; this dissociation rate is too rapid to measure in the presence of bound inducer molecules. Calculations assuming that the DNA-protein association rate is unaffected by sugar binding suggest that the rate of dissociation of DNA from repressor containing 4 bound inducer molecules may be as high as 40 S-1. Determination of the apparent association rate for inducer binding to operator DNA fragments indicates that DNA molecules do dissociate from the protein . operator . inducer complex at intermediate stages during the sugar binding reaction. The exact point at which dissociation occurs at a complex function of the concentrations of operator and inducer and the relationship between the rates for sugar binding and for operator fragment dissociation which are comparable for some of the protein species.

The crystal structure of sodium guanylyl-3′,5′-cytidine (GpC) nonahydrate has been determined by X-ray diffraction procedures and refined to an R value of 0.054. GpC crystallizes with four molecules per monoclinic unit cell, space group C2, with cell dimensions: a = 21.460, b = 16.297, c = 9.332 A and β = 90.54 ° . Two molecules of GpC related by the 2-fold axis of the crystal form a small segment of right-handed, anti-parallel double-helical RNA in the crystal. Guanine is paired to cytosine through three hydrogen bonds of lengths 2.91, 2.95 and 2.86 A. The bases along each strand are heavily stacked at a distance of about 3.4 A. The fragments form skewed flattened rods within the lattice by the inter-molecular stacking of guanines with each other and the stacking of cytosine with the guanosine Ol′atom. The sodium cations are bound only to the ionized phosphate groups in this structure and exhibit face-sharing octahedral co-ordination. The sodium cations serve to bridge the rods of GpC fragments and organize them into sheets within the crystal. There are 18 water molecules per double-helical fragment which are all part of the first co-ordination shell of nitrogen, oxygen or sodium atoms.

The crystal structure of sodium adenylyl-3′,5′-uridine (ApU) hexahydrate has been determined by X-ray diffraction procedures and refined to an R factor of 0.057. ApU crystallizes with two molecules per asymmetric unit in a monoclinic unit cell, space group P21, with cell dimensions: a = 18.025, b = 17.501, c = 9.677 A and β = 99.45 ° . The two independent molecules of ApU form a small segment of right-handed antiparallel double-helical RNA in the crystal, with Watson-Crick base-pairing between adenine and uracil. This is the first time that this Watson-Crick base-pair has been seen unambiguously at atomic resolution and it is also the first time that a nucleic acid fragment with double-helical symmetry has been seen at atomic resolution. The distance between the C1′ atoma of the adenine-uracil base-pair is slightly shorter than the analogous distance seen in guanine-cytosine base-pairs. The bases in each strand are heavily stacked. One sodium cation binds to the phosphates, as expected; however, the other sodium cation binds on the dyad axis in the minor groove of the double helix. It is co-ordinated directly to the two uracil carbonyl groups which protrude into the minor groove and is shielded from the nearest phosphates by a shell of water. This binding appears to be sequence-specific for ApU. One of the adenines also forms a pair of hydrogen bonds to a nearby ribose, utilizing N6 and N7. The 12 water molecules per double-helical fragment are all part of the first co-ordination shell. The ions and the symmetry of the double-helical fragment are the major organizing elements of the solvent region.

The structure of a complex between Eco RI endonuclease and a cognate oligonucleotide shows that sequence specificity is mediated by 12 protein-DNA hydrogen bonds. These interactions discriminate the Eco RI recognition site from all other sequences because any base substitution would rupture at least one of these hydrogen bonds.

Blunt end ligation with an EcoRI/ octanucleotide “linker” was used to construct a plasmid containing chemically synthesized lac operator DNA. This plasmid renders the host bacterium constitutive for β-galactosidase.

The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.

The dinucleoside phosphates ApU and GpC form right-handed anti-parallel double helical fragments within their crystal lattices. Using a least squares procedure, we have generated the extended double helices which these fragments represent. ApU corresponds to a double helix with 11.9 residues per turn and a pitch of 28. 1A. The GpC double helix has 10.4 residues per turn and a pitch of 26. 9A.

Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding VM values of 3.1 A3/dal and 2.8 A3/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice.

Under standard conditions, Eco RI endonuclease uniquely recognizes the inverted repeat GAATTC. However, this specificity breaks down under non-standard conditions into what has been termed Eco RI* specificity, wherein many other sequences are recognized. We show here that the hydrolysis rates at all known Eco RI* sites can be summarized by the hierarchies: G much greater than A greater than T much greater than C at the first position, A much greater than [G,C] much greater than T at the second and third position, and the corresponding complements at the last three positions. This is consistent with a recognition model which assumes that there are two specific hydrogen bonds per base pair under standard conditions. One or more of these are randomly replaced by water under Eco RI* conditions and the position of a sequence within the appropriate hierarchy is primarily determined by the number of retained hydrogen bonds. These retained hydrogen bonds are common recognition features that can be identified by examining the DNA. The recognition points thereby identified for Eco RI all fall within the major groove of the DNA.

EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.