The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organizati... more The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.
Deciphering the X-ray crystal structures of serine protease inhibitors (serpins) and serpin compl... more Deciphering the X-ray crystal structures of serine protease inhibitors (serpins) and serpin complexes has been an integral part of understanding serpin function and inhibitory mechanisms. In addition, high-resolution structural information of serpins derived from the three domains of life (bacteria, archaea, and eukaryotic) and viruses has provided valuable insights into the hereditary and evolutionary history of this unique superfamily of proteins. This chapter will provide an overview of the predominant biophysical method that has yielded this information, X-ray crystallography. In addition, details of up-and-coming methods, such as neutron crystallography, cryo-electron microscopy, and small- and wide-angle solution scattering, and their potential applications to serpin structural biology will be briefly discussed. As serpins remain important both biologically and medicinally, the information provided in this chapter will aid in future experiments to expand our knowledge of this family of proteins.
Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of canc... more Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) ...
Acta crystallographica. Section F, Structural biology communications, Jun 1, 2018
Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein d... more Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X-ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40-80 µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7 Å resolution and were indexed in space group P2, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0 Å, β = 104.2°. These preliminary results provide the necessary framework for time-resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.
Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydrat... more Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO/HCO. Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W') is observed next to the previously observed intermediate water W, and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH o...
The microenvironment within a solid tumor is usually heterogeneous with certain regions being aci... more The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme a...
European journal of medicinal chemistry, Jan 26, 2017
Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibito... more Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibitors for human carbonic anhydrase hCA IX and XII, with one such compound (SLC-0111/U-F) currently in clinical trials (clinical trials.gov, NCT02215850). In this study, the crystal structures of both hCA II (off-target) and an hCA IX-mimic (target) in complex with selected USBs (U-CH3, U-F, and U-NO2), at resolutions of 1.9 Å or better, are presented, and demonstrate differences in the binding modes within the two isoforms. The presence of residue Phe 131 in hCA II causes steric hindrance (U-CH3, 1765 nM; U-F, 960 nM; U-NO2, 15 nM) whereas in hCA IX (U-CH3, 7 nM; U-F, 45 nM; U-NO2, 1 nM) and hCA XII (U-CH3, 6 nM; U-F, 4 nM; U-NO2, 6 nM), 131 is a Val and Ala, respectively, allows for more favorable binding. Our results provide insight into the mechanism of USB selective inhibition and useful information for structural design and drug development, including synthesis of hybrid USB compounds ...
Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors... more Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anti-cancer agents with some entering Phase I clinical trials. hCA IX is a transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical/biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible and its catalytic efficiency is suggested to be correlated directly with its stability between 3.0<pH<8.0 but not at pH>8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low pH tumor microenvironments, and may be applicable to d...
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculi... more The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and i...
The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organizati... more The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.
Deciphering the X-ray crystal structures of serine protease inhibitors (serpins) and serpin compl... more Deciphering the X-ray crystal structures of serine protease inhibitors (serpins) and serpin complexes has been an integral part of understanding serpin function and inhibitory mechanisms. In addition, high-resolution structural information of serpins derived from the three domains of life (bacteria, archaea, and eukaryotic) and viruses has provided valuable insights into the hereditary and evolutionary history of this unique superfamily of proteins. This chapter will provide an overview of the predominant biophysical method that has yielded this information, X-ray crystallography. In addition, details of up-and-coming methods, such as neutron crystallography, cryo-electron microscopy, and small- and wide-angle solution scattering, and their potential applications to serpin structural biology will be briefly discussed. As serpins remain important both biologically and medicinally, the information provided in this chapter will aid in future experiments to expand our knowledge of this family of proteins.
Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of canc... more Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) ...
Acta crystallographica. Section F, Structural biology communications, Jun 1, 2018
Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein d... more Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X-ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40-80 µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7 Å resolution and were indexed in space group P2, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0 Å, β = 104.2°. These preliminary results provide the necessary framework for time-resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.
Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydrat... more Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO/HCO. Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W') is observed next to the previously observed intermediate water W, and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH o...
The microenvironment within a solid tumor is usually heterogeneous with certain regions being aci... more The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme a...
European journal of medicinal chemistry, Jan 26, 2017
Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibito... more Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibitors for human carbonic anhydrase hCA IX and XII, with one such compound (SLC-0111/U-F) currently in clinical trials (clinical trials.gov, NCT02215850). In this study, the crystal structures of both hCA II (off-target) and an hCA IX-mimic (target) in complex with selected USBs (U-CH3, U-F, and U-NO2), at resolutions of 1.9 Å or better, are presented, and demonstrate differences in the binding modes within the two isoforms. The presence of residue Phe 131 in hCA II causes steric hindrance (U-CH3, 1765 nM; U-F, 960 nM; U-NO2, 15 nM) whereas in hCA IX (U-CH3, 7 nM; U-F, 45 nM; U-NO2, 1 nM) and hCA XII (U-CH3, 6 nM; U-F, 4 nM; U-NO2, 6 nM), 131 is a Val and Ala, respectively, allows for more favorable binding. Our results provide insight into the mechanism of USB selective inhibition and useful information for structural design and drug development, including synthesis of hybrid USB compounds ...
Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors... more Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anti-cancer agents with some entering Phase I clinical trials. hCA IX is a transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical/biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible and its catalytic efficiency is suggested to be correlated directly with its stability between 3.0<pH<8.0 but not at pH>8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low pH tumor microenvironments, and may be applicable to d...
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculi... more The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and i...
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