Short oxygen-halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter-and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short COX⅐⅐⅐OOY interaction (COX is a carbon-bonded chlorine, bromine, or iodine, and OOY is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X⅐⅐⅐O distance is less than or equal to the sums of the respective van der Waals radii (3.27 Å for Cl⅐⅐⅐O, 3.37Å for Br⅐⅐⅐O, and 3.50 Å for I⅐⅐⅐O) and can conform to the geometry seen in small molecules, with the COX⅐⅐⅐O angle Ϸ165°(consistent with a strong directional polarization of the halogen) and the X⅐⅐⅐OOY angle Ϸ120°. Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology. molecular folding ͉ molecular recognition ͉ molecular design T wo recent biomolecular single-crystal structures, a fourstranded DNA Holliday junction (1) and an ultrahighresolution structure (0.66 Å) of the enzyme aldose reductase complex with a halogenated inhibitor (2), revealed unusually short Br⅐⅐⅐O contacts [Ϸ3.0 Å, or Ϸ12% shorter than the sum of their van der Waals radii (R vdW )]. The atypical contact in the enzyme complex was attributed to an electrostatic interaction between the polarized bromine and the lone pair electrons of the oxygen atom of a neighboring threonine side chain (3). Short halogen-oxygen interactions are not in themselves new: The chemist Odd Hassel (4) had earlier described Br⅐⅐⅐O distances as short as 2.7 Å (Ϸ20% shorter than R vdW ) in crystals of Br 2 with various organic compounds.These short contacts, originally called charge-transfer bonds, were attributed to the transfer of negative charge from an oxygen, nitrogen, or sulfur (a Lewis base) to a polarizable halogen (a Lewis acid) (5, 6). They are now referred to as halogen bonds (Fig. 1) by analogy to classical hydrogen bonds with which they share numerous properties (6) and are currently being exploited to control the crystallization of organic compounds in the design of new materials (7) as well as in supramolecular chemistry (6). Extensive surveys of structures in the Cambridge Structural Database (8-10) coupled with ab initio calculations (10) have characterized the geometry of halogen bonds in small molecules and show that the interaction is primarily electrostatic, with contributions from polar...

The halogen bond, a noncovalent interaction involving polarizable chlorine, bromine, or iodine molecular substituents, is now being exploited to control the assembly of small molecules in the design of supramolecular complexes and new materials. We demonstrate that a halogen bond formed between a brominated uracil and phosphate oxygen can be engineered to direct the conformation of a biological molecule, in this case to define the conformational isomer of a four-stranded DNA junction when placed in direct competition against a classic hydrogen bond. As a result, this bromine interaction is estimated to be Ϸ2-5 kcal/mol stronger than the analogous hydrogen bond in this environment, depending on the geometry of the halogen bond. This study helps to establish halogen bonding as a potential tool for the rational design and construction of molecular materials with DNA and other biological macromolecules.biomolecular engineering ͉ DNA structure ͉ molecular interactions H alogen bonds have recently seen a resurgence of interest as a tool for ''bottom-up'' molecular design. Chlorines, bromines, and iodines in organic and inorganic compounds are known to polarize along their covalent bonds to generate an electropositive crown; the halogen thus acts as a Lewis acid to pair with Lewis bases, including oxygens and nitrogens. These electrostatic pairs, originally called charge-transfer bonds (1), are now known as halogen bonds (X-bonds), recognizing their similarities to hydrogen bonds (H-bonds) in their strength and directionality (2). In chemistry, X-bonds are being exploited in the design and engineering of supramolecular assemblies (3) and molecular crystals (for review, see ref. 4), with an iodine X-bond estimated to be Ϸ3.5 kcal/mol more stable than an O-H⅐⅐⅐O H-bond in organic crystals (5).The X-bond, however, has not generally been a part of the biologist's lexicon. Although halogens are widely used in drug design and to probe molecular interactions, X-bonds have only recently been recognized as a distinct interaction in ligand recognition and molecular folding and in the assembly of proteins and nucleic acids (6, 7). With the growing application of biological molecules (biomolecule), particularly nucleic acids (for review, see ref. 8), in the design of nanomechanical devices, we ask here whether specific X-bonds can be engineered to direct conformational switching in a biomolecule.To compare X-and H-bonds in the complex environment of a biomolecule, we have designed a crystallographic assay to determine whether an intramolecular X-bond could be engineered to direct the conformational isomerization of a DNA Holliday junction by competing an X-bond against a classic H-bond and, consequently, we are able to compare the stabilization energies afforded by these two types of interactions. The stacked-X form of the DNA Holliday junction (Fig. 1), seen in high-salt solutions (9) and in crystal structures (10 -12), is a simple and well controlled biomolecular assay system that can isomerize between two nearly isoenergetic an...

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Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that mediate the transport of nucleosides, nucleobases, and therapeutic analogs. The best-characterized ENTs are the human transporters hENT1 and hENT2. However, non-mammalian eukaryotic ENTs have also been studied (e.g., yeast, parasitic protozoa). ENTs are major pharmaceutical targets responsible for modulating the efficacy of more than 30 approved drugs. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. This review highlights findings on the characterization of ENTs by surveying studies on genetics, permeant and inhibitor interactions, mutagenesis, and structural models of ENT function.

Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the a5 helix was released into the MOM, the remaining interface with a2, a3, and a4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel a9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, a9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by a9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by a9 dimerization at the rim.

Quinoxalinedione compounds such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) are the most commonly used alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists. However, we find that in the presence of transmembrane AMPA receptor regulatory proteins (TARPs), which are AMPA receptor auxiliary subunits, CNQX acts as a partial agonist. CNQX induced small depolarizing currents in neurons of the central nervous system, and reconstitution of this agonist activity required coexpression of TARPs. A crystal structure of CNQX bound to the TARP-less AMPA receptor ligand-binding domain showed that, although CNQX induces partial domain closure, this movement is not transduced into linker separation, suggesting that TARPs may increase agonist efficacy by strengthening the coupling between domain closure and channel opening. Our results demonstrate that the presence of an auxiliary subunit can determine whether a compound functions as an agonist or antagonist.

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

Structures of the DNA sequences d(CCGGCGCCGG) and d(CCAGTACbr(5)UGG) are presented here as four-way Holliday junctions in their compact stacked-X forms, with antiparallel alignment of the DNA strands. Thus, the ACC-trinucleotide motif, previously identified as important for stabilizing the junction, is now extended to PuCPy, where Pu is either an adenine or guanine, and Py is either a cytosine, 5-methylcytosine, or 5-bromouracil but not thymine nucleotide. We see that both sequence and base substituents affect the geometry of the junction in terms of the interduplex angle as well as the previously defined conformational variables, J(roll) (the rotation of the stacked duplexes about their respective helical axis) and J(slide) (the translational displacement of the stacked duplexes along their respective helical axis). The structures of the GCC and parent ACC containing junctions fall into a distinct conformational class that is relatively undistorted in terms of J(slide) and J(roll), with interduplex angles of 40-43 degrees. The substituted ACbr(5)U structure, however, is more akin to that of the distorted methylated ACm(5)C containing junction, with J(slide) (>or=2.3 A) and a similar J(roll) (164 degrees) opening the major groove-side of the junction, but shows a reduced interduplex angle. In contrast, the analogous d(CCAGTACTGG) sequence has to date been crystallized only as resolved B-DNA duplexes. This suggests that there is an electronic effect of substituents at the pyrimidine Py position on the stability of four-stranded junctions. The single-crystal structures presented here, therefore, show how sequence affects the detailed geometry, and subsequently, the associated stability and conformational dynamics of the Holliday junction.