Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD) to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

Role of Trp19 and Tyr200 in catalysis by the γ-class carbonic anhydrase from Methanosarcina thermophila.

Although widely distributed in Nature, only two γ class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state k(cat)/K(m) and k(cat) values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in k(cat). Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ~2-fold for Y200F. The pK(a) values for the proton donor decreased 1-2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62-64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO(2)/bicarbonate interconversion, and the proton transfer step of catalysis.

Sequestration of carbon dioxide by the hydrophobic pocket of the carbonic anhydrases.

The interaction between carbon dioxide (CO(2)) and the alpha-class carbonic anhydrase, human CA 2 (HCA2) exists for only a short period due to the rapid catalytic turnover by this enzyme. The fleeting nature of this interaction has led to difficulties in its direct analysis, with previous studies placing the CO(2) in the hydrophobic pocket of HCA2's active site. A more precise location was determined via the crystal structure of CO(2) trapped in both wild-type (holo) and zinc-free (apo) HCA2. This provided a detailed description of the means by which CO(2) is held and orientated for optimal catalysis. This information can be extended to the beta and gamma class enzymes to help elucidate the binding mode of CO(2) in these enzymes.

Structure and catalysis by carbonic anhydrase II: role of active-site tryptophan 5.

The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1µs(-1), determined by exchange of (18)O between CO(2) and water measured by mass spectrometry. The apparent values of the pK(a) of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO(2) and bicarbonate.

Structural and kinetic study of the extended active site for proton transfer in human carbonic anhydrase II.

The catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer from a zinc-bound water molecule to the proton shuttle His64. This proton transfer occurs along a hydrogen-bonded water network, leading to the proton shuttle residue His64, which in turn transfers the proton to bulk solvent. The side chain of His64 occupies two conformations in wild-type HCA II, pointing inward toward the zinc or outward toward bulk solvent. Previously, several studies have examined the roles of residues of the active site cavity that interact with the solvent-mediated hydrogen-bonded network between His64 and the zinc-bound water. Here these studies are extended to examine the effects on proton transfer by mutation at Lys170 (to Ala, Asp, Glu, and His), a residue located near the side chain of His64 but over 15 A away from the active site zinc. In all four variants, His64 is observed in the inward conformation associated with a decrease in the pK(a) of His64 by as much as 1.0 unit and an increase in the rate constant for proton transfer to as much as 4 micros(-1), approximately 5-fold larger than wild-type HCA II. The results show a significant extension of the effective active site of HCA II from the zinc-bound water at the base of the conical cavity in the enzyme to Lys170 near the rim of the cavity. These data emphasize that the active site of HCA II is extended to include residues that, at first glance, appear to be too far from the zinc to exert any catalytic effects.

Neutron structure of human carbonic anhydrase II: implications for proton transfer.

Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 A resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D(2)O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.

Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinic P2(1) with a doubled a axis.

The crystal structure of human carbonic anhydrase II in the monoclinic P2(1) space group with a doubled a axis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbins et al. (2010), Acta Cryst. D66, 628-634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated by x' = x +/- 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit.

Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axis.

The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4 A resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of (1/2) along a, but one of the molecules has two alternate positions related by a rotation of approximately 2 degrees. This rotation axis is located near the edge of the central beta-sheet, causing a maximum distance disparity of 1.7 A between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final R(cryst) and R(free) values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h-odd data.

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase.

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.

Entrapment of carbon dioxide in the active site of carbonic anhydrase II.

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO(2)-pressurized, cryo-cooled crystals to capture the first step of CO(2) hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO(2), for both the holo and the apo (without zinc) enzyme to 1.1A resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO(2) and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO(2) in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO(2). This method may also have a much broader implication for the study of other enzymes for which CO(2) is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

Structural and kinetic study of differences between human and Escherichia coli manganese superoxide dismutases.

Human manganese superoxide dismutase (MnSOD) is characterized by a product inhibition stronger than that observed in bacterial forms of MnSOD. Previous studies show that the conserved, active-site residue Tyr34 mediates product inhibition; however, the protein environment of Tyr34 is different in human and Escherichia coli MnSOD. We have prepared two site-specific mutants of human MnSOD with replacements of Phe66 with Ala and Leu (F66A and F66L, respectively), altering the surroundings of Tyr34. Pulse radiolysis was used to generate superoxide, and measurements of catalysis were taken in single-turnover experiments by observing the visible absorbance of species of MnSOD and under catalytic conditions observing the absorbance of superoxide. The mutation of Phe66 to Leu resulted in a mutant of human MnSOD with weakened product inhibition resembling that of E. coli MnSOD. Moreover, the mechanism of this weakened product inhibition was similar to that in E. coli MnSOD, specifically a decrease in the rate constant for the oxidative addition of superoxide to Mn2+MnSOD leading to the formation of the peroxide-inhibited enzyme. In addition, the crystal structures of both mutants have been determined and compared to those of wild-type human and E. coli MnSOD. The crystallographic data suggest that the solvent structure and its mobility as well as side chain conformations may affect the extent of product inhibition. These data emphasize the role of residue 66 in catalysis and inhibition and provide a structural explanation for differences in catalytic properties between human and certain bacterial forms of MnSOD.