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 zincmetalloenzyme human carbonic anhydrase II, the binding of substrate CO 2 , for both the holo and the apo (without zinc) enzyme to 1.1 Å 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.Since their discovery (2), the carbonic anhydrases (CAs) 3 have been extensively studied because of their important physiological functions in all kingdoms of life (3). This family of enzymes is broadly comprised of three well studied, structurally distinct families (␣, ␤, and ␥) of mostly zinc-metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate (3, 4). More recently there have been other more distinct CAs characterized, such as a cadmium ␤ class-mimic CA (5). However, all appear to share the same overall catalytic mechanism composed of two independent stages, shown in Equations 1 and 2, an example of a ping-pong mechanism (6, 7). In the hydration direction, the first stage is the conversion of CO 2 into bicarbonate via a nucleophilic attack on CO 2 by the reactive zinc-bound hydroxide. The resultant bicarbonate is then displaced from the zinc by a water molecule (Reaction 1).The second stage is the transfer of a proton from the zincbound water to bulk solvent to regenerate the zinc-bound hydroxide (Reaction 2). Here B is a proton acceptor in solution or a residue of the enzyme itself.For hCAII (␣ class CA), this reaction is facilitated by a solvent molecule with a pK a near 7 that is directly coordinated to the zinc (6). This centrally located zinc exhibits a tetrahedral configuration with three histidines (His-94, His-96, and His-119) and either a water or a hydroxide molecule. The active site cavity can be loosely described as being conical in shape having a 15 Å diameter entrance that tapers into the center of the enzyme. The cavity is partitioned into two very different environments. On one side of the zinc, deep within the active site, lies a cluster of hydrophobic amino acids (namely , whereas on the other side of the zinc, leading out of the active site to the bulk solvent, the surface is lined with hydrophilic amino acids (namely Tyr-7, Asn-62...

The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 Å resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 Å. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO2 in the enzyme-substrate complex, a weak binding that enhances kcat for the conversion of CO2 into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pKa of the zinc-bound water and to promote proton transfer in catalysis.

The V617F mutation in the Jak2 pseudokinase domain causes myeloproliferative neoplasms, and the equivalent mutation in Jak1 (V658F) is found in T-cell leukemias. Crystal structures of wild type and V658F mutant human Jak1 pseudokinase reveal a conformational switch that remodels a linker segment encoded by exon 12, which is also a site of mutations in Jak2. This switch is required for V617F-mediated Jak2 activation, and possibly for physiologic Jak activation.

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.

on the conformation and the stability of-and-crystallins.- ,-and-crystallins are the main components of mammalian eye lenses and their structural and associative properties are responsible for lens transparency. are monomers (21 kDa, up to 80% sequence identity), whereas are large hetero-oligomers of about 800kDa. The C-terminal domain of belongs to the ubiquitous superfamily of sHSPs (small heat shock proteins): upon stress, they are able to incorporate the non-native proteins to prevent their aggregation. High-pressure experiments performed with-crystallins have shown a partially reversible change in size from 2 to 3kb at room temperature, and this effect was enhanced by the combination of temperature and pressure. In the case of-crystallins, pressure and temperature needed to be combined with pH, and the results depend upon the different itself. Crystallins are known to be exceptionally stable in vivo since they are synthesised to last for life. They therefore represent an extreme case of stability versus unfolding and these results have shown that these proteins (mainly beta strands) are also stable upon pressure. Until recently, only two crystal structures of small proteins at high pressure below 200 MPa generated in a Be cell were published [1,2]. The lack of structural data at high pressure was due mainly to the cumulated complexities of high-pressure containment and crystallography. A technical breakthrough was achieved with a setup at the ESRF ID30/ID27 beamline combining a diamond anvil cell, ultra-short wavelength (0.33 Å) X-rays from undulators and a large imaging plate [3]. The accessible pressure range was increased by nearly one order of magnitude. The quality of diffraction data collected under high pressure achieved usual standards. We will present the technical advances as well as scientific results that we have obtained. In particular, scientific results will focus on the first crystal structure of a complex macromolecular assembly under high pressure, the Cowpea Mosaic Virus capsid at 330 MPa [4], demonstrating that high pressure macromolecular crystallography can now be considered as a mature and general technique. The flash cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and, in certain cases unsuccessful. Recently we have developed a novel method to cryocool protein crystals without the need for penetrative cryoprotectants. In the new method, each protein crystal is pressurized up to 200 MPa (2000 atm) in He gas at 10˚C10˚C. The crystal is then cyrocooled under pressure and the pressure was released while the crystal is kept cooled. Results are presented for two proteins that have been flash-cooled at ambient pressure and pressure-cooled, in all case without penetrating cryoprotectants. For glucose isomerase, the flash-cooled crystal diffracted to only 5.0 Å and mosaicity could ...

A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, no examples that correlate protein function with progressive subangstrom structural perturbations have thus far been presented. To elucidate this relationship, we have investigated a fluorescent protein, citrine, as a model system under high-pressure perturbation. The protein has been compressed to produce deformations of its chromophore by applying a high-pressure cryocooling technique. A closely spaced series of x-ray crystallographic structures reveals that the chromophore undergoes a progressive deformation of up to 0.8 Å at an applied pressure of 500 MPa. It is experimentally demonstrated that the structural motion is directly correlated with the progressive fluorescence shift of citrine from yellow to green under these conditions. This protein is therefore highly sensitive to subangstrom deformations and its function must be understood at the subangstrom level. These results have significant implications for protein function prediction and biomolecule design and engineering, because they suggest methods to tune protein function by modification of the protein scaffold.fluorescence ͉ high-pressure x-ray crystallography ͉ protein engineering ͉ protein structure-function ͉ yellow fluorescent protein

Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.

High-pressure methods for solving protein structures by X-ray crystallography and NMR are maturing. These techniques are beginning to impact our understanding of thermodynamic and structural features that define not only the protein's native conformation, but also the higher free energy conformations. The ability of high-pressure methods to visualize these mostly unexplored conformations provides new insight into protein function and dynamics. In this review, we begin with a historical discussion of high-pressure structural studies, with an eye toward early results that paved the way to mapping the multiple conformations of proteins. This is followed by an examination of several recent studies that emphasize different strengths and uses of high-pressure structural studies, ranging from basic thermodynamics to the suggestion of high-pressure structural methods as a tool for protein engineering.