X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO2, producing
Röntgen discovered a form of radiation in 1895 while analyzing the range of cathode rays in vacuum tubes. He termed this radiation X-rays and determined that the permeability of an object to such radiation directly correlates to its density [
The growth of protein crystals dates back as early as 1840 with the observation of hemoglobin crystals in blood samples [
Nearly a decade after the first observation of X-ray diffraction, interest in obtaining neutron diffraction from single crystals increased. X-ray crystallography requires electrons in the sample to interact with the incoming X-ray beam to generate a diffraction pattern. The scattering factor of an atom is the likelihood of a diffraction event occurring and is dependent on how many electrons are in the atom. Electron rich atoms have a high scattering factor, meaning they are easily distinguishable from the diffraction pattern [
As interest in protein structure and function continued to grow, the need for a database of crystal structures led to the development of the Protein Data Bank (PDB, rcsb.org) in 1971. The PDB was started with 7 depositions, including the structures of myoglobin and hemoglobin, and has grown to over 125,000 structures to date [
In the 1920s, two theories were proposed concerning the transport of CO2 in the blood. The most common hypothesis was termed the
CA I, CA II, and CA III were confirmed as unique CA isoforms through sequence comparison and gene mapping [
Since the discovery of mammalian CAs in 1932, 7 genetically unique families have been identified:
The
The
To date, crystal structures have only been determined for the
Crystal structures of catalytically active human CA isoforms.
| | | | | | | | | | |
---|---|---|---|---|---|---|---|---|---|---|
| 24 | 703 | 5 | 10 | 1 | 2 | 6 | 16 | 13 | 2 |
| ||||||||||
| 2CAB | 3KS3 | 1Z93 | 1ZNC | 3FE4 | 3MDZ | 5DVX | 1JCZ | 3D0N | 4LU3 |
| ||||||||||
| 2.0 | 0.9 | 2.1 | 2.8 | 1.9 | 2.3 | 1.6 | 1.6 | 1.6 | 2.0 |
| ||||||||||
| P212121 | P21 | P65 | C2 | P212121 | C2221 | P212121 | C2 | P21 | P41212 |
| ||||||||||
| | | | | | | | | | |
| ||||||||||
| Monomer | Monomer | Monomer | Monomer | Monomer | Monomer | Monomer | Dimer | Monomer | Monomer |
| ||||||||||
| 61 | - | 58 | 34 | 35 | 56 | 34 | 36 | 61 | 37 |
| ||||||||||
| 0.9 | - | 0.8 | 1.3 | 1.4 | 0.6 | 1.4 | 1.2 | 0.8 | 1.2 |
To date, no crystal structures of CA VA, CAVB, CA X, or CA XI have been deposited in the PDB.
The first
The first crystal structure of a
Fifteen
Crystals structures of
CAs are some of the most catalytically efficient enzymes known with CA II exhibiting a turnover rate of 1.1
The full mechanism was first reported in 1988 by Silverman and Lindskog where they described the interworking of the
The second, rate limiting step is the regeneration of the zinc-bound hydroxyl via proton transfer from the zinc-bound water to bulk solvent [
Due to the highly efficient turnover rate of CA II and the limited solubility of CO2 in solution, the binding sites of substrates for CAs remained unknown until hypothesized via molecular dynamics (MD). Using free energy perturbation simulations, Liang and Lipscomb identified three potential CO2 binding sites in the hydrophobic region of the active site. One site was identified as the potential catalytic site and two other binding locations likely sites for CO2 replenishment [
The first CA structures confirming the MD predictions were obtained via cryocooling holo- and apo-CA II under 15 atm of pressurized CO2 [
Similarly, the binding site of
To further investigate
The second step of the
The initial MD predictions for the proton wire were performed in 2006 by Fisher et al. Here, they performed a simplified MD simulation, modeling the CA II structure coordinates with 220 locations of water molecules inside a solvated cubic simulation box [
Subsequent MD simulations at a higher degree of complexity were performed in an attempt to model the experimentally observed rates of CAs. However, in the early 2000s, many of these experiments produced varying results due to differences in MD methodology [
First, the zinc-bound H2O interacts with W1 to form a Zundel cation (
This MS-EVB MD simulation also accurately predicts H64 proton acceptance and donation. When H64 is in the “out” conformation the free energy barrier for proton transfer is 14.6 ± 0.4 kcal/mol [
To confirm the MS-EVB MD simulations, further experiments were conducted to mutate H64 and confirm the importance of proton transfer. H64 was mutated in CA II to Ala and a combination of kinetic, structure, and MD experiments were conducted to determine the effect [
While X-ray crystallography is utilized to determine the solvent molecule positions throughout the structure, the methodology is unable to determine their orientation. The clustered water orientations are needed to validate the proton wire as this mechanism largely depends on distinct orientations of the waters, allowing them to span a network of hydrogen bonds. Recent work done by Fisher et al. performed neutron crystallography on CA II at varying pHs to discover the orientations of water in the proton wire [
Multiple
Subsequent studies revealed that heterocyclic sulfonamides exhibited >1,000-fold increase in affinity in comparison to benzene derivatives with a general increase in activity as the acidity of the compound increased [
Glaucoma is a disease characterized by high intraocular pressure (IOP) and loss of vision. An increase in IOP is most often associated with the retention of aqueous humor, a liquid between the cornea and lens, which is caused by an absence or decrease in humor drainage. A main component of this aqueous humor is sodium bicarbonate, the secretion of which is controlled by CA activity in the uvea of the eye. The inhibition of CA II, CA IV, and CA XII has been shown to decrease the secretion of humor, resulting in decreased IOP. Therefore, CA inhibitors are used in conjunction with adrenergic agonists or antagonists for the treatment of glaucoma [
The first CAIs used to treat glaucoma include acetazolamide, methazolamide, ethoxzolamide, and dichlorphenamide. However, these compounds act systemically, inhibiting multiple CA isoforms, and have been shown to cause unwanted side effects such as fatigue, abnormal tingling, and kidney stones [
Recent developments in antiglaucoma inhibitors use the tail method with an aromatic sulfonamide scaffold to which amino, hydroxyl, or nitrate ester groups are added to increase solubility. These novel compounds have been tested as topical agents in animals and show good solubility and inhibitory effects, resulting in prolonged decreased IOP. In addition, compounds that contain a NO donating moiety can also aid in vasodilation and humor secretion, supplying more blood to the optic nerve and further decreasing IOP. These so-called “hybrid drugs” were also tested in animals and exhibited a greater reduction in IOP than either brinzolamide or dorzolamide, representing a promising class of lead compounds for antiglaucoma drug development [
Edema is a condition characterized by the retention of fluid, often exhibited as a result of heart failure. Heart failure can decrease blood flow to the kidney, impacting filtration rates and decreasing the secretion of water. CA II, CA IV, CA XII, and CA XIV promote pH homeostasis and bicarbonate resorption in the kidney. CAIs have been proven to act as diuretics by inhibiting the exchange of protons and sodium ions, increasing the concentration of excreted sodium, which is accompanied by movement of water to act as a diluent [
Epilepsy is a condition characterized by abnormal brain activity and seizures. It is hypothesized that CA is involved in the secretion of bicarbonate-rich cerebrospinal fluid, similar to CA function in the secretion of aqueous humor in the eye [
Classic CAIs such as acetazolamide and methazolamide have been clinically used as antiepileptics, but have more recently been replaced by topiramate [
Significant weight loss has been observed as a side effect of the antiepileptic CAIs topiramate and zonisamide. CA V research is therefore focused on the possibility of a link between CA V inhibition and weight loss. CA V is expressed in the mitochondria as two forms: Vb exhibits wide tissue distribution whereas Va is limited to liver tissue. CA V provides bicarbonate to serve as a substrate for pyruvate carboxylate in gluconeogenesis and oxaloacetate production in lipogenesis. The inhibition of CA V is therefore expected to induce weight loss by depleting a source of bicarbonate and decreasing fatty acid synthesis [
CA IX overexpression has been observed in several cancer types including lung, renal, brain, colon, pancreatic, liver, breast, esophageal, ovarian, and skin cancer. In contrast, CA IX expression is limited to the GI tract in healthy tissue [
CA IX activity is hypothesized to be critical for the regulation of pH in cancer cells that must thrive in an acidic tumor microenvironment [
As CA IX is a membrane-bound isoform with an extracellular catalytic domain, CAI selectivity can be enhanced by designing membrane impermeable compounds to prevent the off-target binding of cytosolic CAs. For example, positively or negatively charged hydrophilic moieties can be added to promote impermeability. However, these properties make it unlikely that such compounds would enter the bloodstream or be developed into a drug to be taken orally. Therefore, inhibitors are designed as prodrugs with hydrophobic moieties that mask the desired, inhibitory substituents until the compound is present in the reductive conditions of a hypoxic environment where it will be hydrolyzed and become an active inhibitor [
Artificial sweeteners have recently been identified as potential CAIs for the development of cancer therapeutics. Saccharin and acesulfame potassium were shown to bind within the CA active site and exhibit binding affinities in the micromolar range [
Although several CAIs are currently in use for the treatment of diseases, these clinically available compounds do not exhibit sufficient isoform selectivity and therefore bind to multiple CA isoforms. Off-target binding can lead to sequestration of the drug, requiring higher doses for treatment and subsequently decreasing efficacy. However, the design of isoform specific CAIs is complicated by the structural homology shared by the catalytically active CAs. X-ray and neutron crystallography are therefore utilized to analyze the binding of inhibitors in the CA active site and guide drug design of isoform specific inhibitors.
Structure-guided drug design is a technique that uses high resolution crystal structures of a molecular target to rationalize the design of high affinity, small molecule inhibitors. This process often begins with high throughput screening of different classes of inhibitors to identify lead compounds that inhibit CA activity. The most promising compounds, which exhibit binding affinities in the nano- to micromolar range, are then studied using X-ray crystallography. The crystal structure complex is analyzed to identify interactions between the compound and target molecule that promote selective binding. New derivatives can then be designed to promote such interactions via the addition of functional groups to the lead compound [
The tail approach, previously described as a method to improve inhibitor solubility, can also be applied in structure-guided drug design. The mapping and comparison of active site residues between isoforms have identified residues unique to the target isozymes. Elongation or derivatization of a compound tail can promote interactions with these isoform unique residues, improving selectivity [
It is important to recognize that the affinity of a CAI is dependent on the free energy of binding (ΔG = ΔH - T ΔS) with both enthalpic and entropic contributions. A ligand loses rotational freedom upon binding, decreasing ΔS. This entropic penalty can be counteracted by enthalpic gains upon the formation of interactions between the ligand and target molecule in addition to increases in entropy as water molecules are displaced from the active site [
The combination of high resolution X-ray and medium resolution neutron crystal structures has led to a thorough characterization of the
Active site residues in CA I- CA XIV.
| | | | | | | | | | | | | | | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P00915 | P00918 | P07451 | P22748 | P35218 | Q9Y2D0 | P23280 | P43166 | P35219 | Q16790 | Q9NS85 | Q866X7 | O43570 | Q8N1Q1 | Q9ULX7 | |
| |||||||||||||||
| |||||||||||||||
| W | W | W | W | C | C | W | W | W | W | W | W | W | W | W |
| Y | Y | Y | Y | W | Y | Y | Y | Y | Y | Y | Y | Y | Y | Y |
| V | N | N | N | T | N | N | N | D | N | T | T | N | S | N |
| H | H | K | H | Y | Y | H | H | H | H | R | R | H | H | H |
| S | A | T | S | S | L | T | S | T | S | H | H | S | S | T |
| H | N | R | M | M | Q | Q | Q | Q | Q | S | S | K | N | Q |
| Q | Q | Q | Q | Q | Q | Q | Q | E | Q | Q | Q | Q | Q | Q |
| A | V | V | V | V | V | V | V | I | V | I | I | V | V | V |
| L | L | I | I | L | L | L | L | I | L | L | L | L | L | L |
| V | V | V | V | V | V | V | V | I | V | V | I | V | V | V |
| L | L | F | L | L | L | L | L | L | L | M | L | L | L | L |
| H | T | T | T | T | T | T | T | I | T | I | T | T | V | T |
| V | V | I | V | V | V | V | V | V | V | A | V | V | V | V |
| W | W | W | W | W | W | W | W | W | W | W | W | W | W | W |
| N | N | N | N | N | N | N | N | N | N | N | N | N | N | N |
| |||||||||||||||
| |||||||||||||||
| |||||||||||||||
| I | L | L | Q | W | W | V | T | T | R | Y | Y | T | S | H |
| N | E | V | L | E | E | S | D | I | T | R | L | N | D | S |
| F | I | R | K | K | K | Q | K | Y | L | E | S | T | R | A |
| L | F | F | V | Y | F | Y | F | I | V | V | L | A | F | L |
| A | V | L | Q | V | A | Q | A | V | L | A | T | S | A | A |
| P | P | P | P | P | P | P | P | P | P | P | P | P | P | P |
| P | P | P | T | P | P | P | P | P | P | P | P | P | P | P |
| Y | L | E | D | T | S | T | S | S | A | Y | S | N | L | Y |
| |||||||||||||||
| |||||||||||||||
| |||||||||||||||
| D | H | E | H | S | T | D | G | E | H | W | W | K | S | H |
| L | D | L | L | P | V | H | L | V | V | V | V | K | F | S |
| Y | F | F | V | V | D | Y | Y | F | S | N | N | Y | F | Y |
| P | P | P | P | S | L | P | P | P | P | S | A | P | P | P |
| I | I | N | V | V | V | A | I | D | A | A | A | S | I | E |
| K | L | K | W | L | L | F | L | C | L | G | G | F | K | L |
| E | R | T | T | Y | H | P | S | E | R | T | T | L | I | D |
| D | D | D | N | D | D | S | D | S | P | K | P | S | D | S |
| W | W | W | K | W | W | Y | W | W | L | Y | F | Y | W | Y |
| S | D | T | N | N | N | S | T | S | R | N | N | D | S | S |
| A | G | K | K | K | E | D | G | D | D | T | S | S | V | S |
| S | Q | K | D | V | L | D | S | G | G | K | R | N | H | E |
| K | K | K | P | K | K | P | K | K | E | K | K | K | K | K |
| G | G | G | E | D | D | G | G | G | G | N | N | G | G | D |
| R | S | E | S | R | L | R | K | S | E | A | A | E | Q | K |
The hydrophobic half of the active site was then further divided into two pockets in which the tails of inhibitors orient. Pocket 1 consists of residues L198, F131, V135, and L204 whereas Pocket 2 contains I91, V121, and F131 (Figures
In 2013, a structural comparison of the binding of all nonredundant inhibitors in complex with CA II was performed. Of the 145 compounds, only 14 were observed to orient toward a region between the hydrophobic and hydrophilic areas of the active site. This region was termed the “selective pocket” due to the variability of residues 67, 69, 91, and 131 between isoforms (Figure
The use of neutron crystallography in conjunction with X-ray crystallography allows the visualization of both “heavy” (non-H) and “light” (H) atoms. It is therefore possible to determine the protonation state of amino acid side chains and inhibitors using joint refinement. For example, the standard CAI acetazolamide exhibits three possible protonation states in solution. Initial solution state NMR studies hypothesized that sulfonamide-based compounds bind in the deprotonated state and interact with catalytic zinc through the sulfonamide nitrogen [
The search for isoform selective CAIs has also led to the discovery of nonsulfonamide-based inhibitors that exhibit unique mechanisms of inhibition, including compounds that anchor to the zinc-bound water and CAIs that bind outside the active site, occluding entrance of substrate. These compounds have the additional benefit of preventing adverse effects in individuals with sulfa allergies [
Compounds that anchor through the zinc-bound water deselect from zinc binding that is well established for sulfonamide-based inhibitors. Therefore, the binding affinity relies primarily on interactions with residues in the active site. In comparison to compounds that bind directly to the catalytic zinc, inhibitors that anchor through the water extend an additional 2.5–3 Å from the zinc, increasing the probability of forming interactions with isoform unique residues in the selective pocket. Examples of such compounds include phenol- and carboxylic-based inhibitors. Both classes anchor to the zinc-bound solvent through the hydroxyl moiety. Phenol-based compounds exhibit binding affinities within the micromolar range and are hypothesized to inhibit activity via obstruction of the CO2 binding site caused by interactions of the phenyl group with hydrophobic residues in the active site [
The majority of isoform unique residues encircle the entrance of the active site. Therefore, compounds that inhibit activity by occlusion of the active site are more likely to interact with these unique residues, improving isoform selectivity of the CAIs. Examples of this class of inhibitors include disaccharides and artificial sweeteners, such as sucrose and sucralose. Interestingly, sucralose inhibits CA activity in the micromolar range whereas sucrose binds but does not inhibit CA activity. Based on the crystal structures, sucralose binds along the hydrophobic region and is predicted to prevent entry of CO2. In contrast, a small opening into the active site exists when sucrose is bound, allowing movement of CO2 [
Bacterial resistance is a growing issue worldwide and pathogenic strains such as
Sulfonamide-based compounds have been shown to inhibit the activity of
CAs are an essential class of enzymes in every class of life, from marine diatoms and bacteria to humans. The crystal structure of CA II was amongst the first structures to be determined and was one of the seven structures that contributed to the development of the PDB. Structural characterization has not only led to the classification of the seven CA families but has also improved the understanding of the catalytic mechanism. X-ray and neutron crystallography studies have identified the binding sites of both substrate/product and elucidated proton transfer through observation of an ordered water network and dual conformations of the proton shuttle residue. These techniques have also driven the design of CAIs as antiglaucoma, antiepileptic, antiobesity, and anticancer therapies in addition to antibiotics.
Although structure-guided drug design has led to several classes of high affinity CAIs, the majority of these compounds still do not exhibit sufficient isoform selectivity to prevent off-target binding. Therefore, interactions between an inhibitor tail and the four residues of the selective pocket do not adequately differentiate between the fifteen human
Our current knowledge of the catalytic mechanism is limited to static crystal structures and a complete understanding of the dynamics of the reaction has yet to be achieved. Insight into the dynamics of the proton wire has been recently expanded by the determination of X-ray crystal structures from crystals under high CO2 pressure. Varying the time of incubation at room temperature prior to data collection provides a method to visualize CO2 release. This study identified an extension of the active site water network termed the entrance conduit waters (EC1, EC2, EC3, EC4, and EC5). Analysis of the alternate conformations of these dynamic water molecules led to the proposal of a restoration of the zinc-bound water following
The development of X-ray free electron laser (XFEL) sources now allows the design of serial femtosecond crystallography (SFX) experiments on a time scale capable of capturing intermediate states of a catalytic mechanism [
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Carrie L. Lomelino is supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under University of Florida Clinical and Translational Science Awards TL1TR001428 and UL1TR001427.