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1.
MAbs ; 12(1): 1792130, 2020.
Article in English | MEDLINE | ID: mdl-32684124

ABSTRACT

Conditionally active COBRA™ (COnditional Bispecific Redirected Activation) T cell engagers are engineered to overcome the limitations of inherently active first-generation T cell engagers, which are unable to discern between tumor and healthy tissues. Designed to be administered as prodrugs, COBRAs target cell surface antigens upon administration, but engage T cells only after they are activated within the tumor microenvironment (TME). This allows COBRAs to be preferentially turned on in tumors while safely remaining inactive in healthy tissue. Here, we describe the development of the COBRA design and the characterization of these conditionally active T cell engagers. Upon administration COBRAs are engineered to bind to tumor-associated antigens (TAAs) and serum albumin (to extend their half-life in circulation), but are inhibited from interacting with the T cell receptor complex signaling molecule CD3. In the TME, a matrix metalloproteinase (MMP)-mediated linker cleavage event occurs within the COBRA construct, which rearranges the molecule, allowing it to co-engage TAAs and CD3, thereby activating T cells against the tumor. COBRAs are conditionally activated through cleavage with MMP9, and once active are highly potent, displaying sub-pM EC50s in T cell killing assays. Studies in tumor-bearing mice demonstrate COBRA administration completely regresses established solid tumor xenografts. These results strongly support the further characterization of the novel COBRA design in preclinical development studies.


Subject(s)
Antibodies, Bispecific , Antigens, Neoplasm , Antineoplastic Agents, Immunological , Immunotherapy , Lymphocyte Activation , Neoplasms, Experimental/therapy , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/genetics , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/immunology , Antineoplastic Agents, Immunological/pharmacology , HT29 Cells , Humans , Jurkat Cells , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasms, Experimental/immunology , Protein Engineering , Xenograft Model Antitumor Assays
2.
PLoS One ; 8(6): e66669, 2013.
Article in English | MEDLINE | ID: mdl-23805258

ABSTRACT

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Calmodulin-Binding Proteins/metabolism , Kinesins/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/genetics , Crystallography, X-Ray , Dimerization , Kinesins/chemistry , Microscopy, Interference , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ultracentrifugation
3.
Proc Natl Acad Sci U S A ; 108(41): 16927-31, 2011 Oct 11.
Article in English | MEDLINE | ID: mdl-21949357

ABSTRACT

An essential regulator of gene transcription, nuclear receptor liver receptor homologue 1 (LRH-1) controls cell differentiation in the developing pancreas and maintains cholesterol homeostasis in adults. Recent genome-wide association studies linked mutations in the LRH-1 gene and its up-stream regulatory regions to development of pancreatic cancer. In this work, we show that LRH-1 transcription is activated up to 30-fold in human pancreatic cancer cells compared to normal pancreatic ductal epithelium. This activation correlates with markedly increased LRH-1 protein expression in human pancreatic ductal adenocarcinomas in vivo. Selective blocking of LRH-1 by receptor specific siRNA significantly inhibits pancreatic cancer cell proliferation in vitro. The inhibition is tracked in part to the attenuation of the receptor's transcriptional targets controlling cell growth, proliferation, and differentiation. Previously, LRH-1 was shown to contribute to formation of intestinal tumors. This study demonstrates the critical involvement of LRH-1 in development and progression of pancreatic cancer, suggesting the LRH-1 receptor as a plausible therapeutic target for treatment of pancreatic ductal adenocarcinomas.


Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/physiopathology , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/physiopathology , Receptors, Cytoplasmic and Nuclear/physiology , Carcinoma, Pancreatic Ductal/genetics , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Epithelium/metabolism , Gene Expression Regulation, Neoplastic , Humans , Models, Biological , Mutation , Pancreas/metabolism , Pancreatic Neoplasms/genetics , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , Signal Transduction
4.
Proc Natl Acad Sci U S A ; 106(20): 8175-9, 2009 May 19.
Article in English | MEDLINE | ID: mdl-19416847

ABSTRACT

Much of the transport, tension, and movement in mitosis depends on kinesins, the ATP-powered microtubule-based motors. We report the crystal structure of a kinesin complex, the mitotic kinesin KCBP bound to its principal regulator KIC. Shown to be a Ca(2+) sensor, KIC works as an allosteric trap. Extensive intermolecular interactions with KIC stabilize kinesin in its ADP-bound conformation. A critical component of the kinesin motile mechanism, called the neck mimic, switches its association from kinesin to KIC, stalling the motor. KIC denies access of the motor to its track by steric interference. Two major features of this regulation, allosteric trapping and steric blocking, are likely to be general for all kinesins.


Subject(s)
Arabidopsis Proteins/chemistry , Calcium-Binding Proteins/chemistry , Calmodulin-Binding Proteins/chemistry , Kinesins/chemistry , Microtubule-Associated Proteins/chemistry , Crystallography, X-Ray , Mitosis , Protein Binding , Protein Conformation
5.
J Struct Biol ; 163(1): 76-83, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18513992

ABSTRACT

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix alpha4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.


Subject(s)
Calmodulin-Binding Proteins/chemistry , Kinesins/chemistry , Microtubules/metabolism , Molecular Motor Proteins/chemistry , Plant Proteins/chemistry , Binding Sites , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/physiology , Crystallography, X-Ray , Kinesins/metabolism , Plant Proteins/physiology , Protein Conformation , Solanum tuberosum
7.
J Mol Biol ; 357(3): 707-17, 2006 Mar 31.
Article in English | MEDLINE | ID: mdl-16469331

ABSTRACT

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.


Subject(s)
Calcium/chemistry , Calcium/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Models, Molecular , Actins/chemistry , Actins/metabolism , Actins/ultrastructure , Humans , Microfilament Proteins/ultrastructure , Protein Structure, Tertiary , Software , Tropomyosin/chemistry , Tropomyosin/metabolism , Tropomyosin/ultrastructure , Troponin/chemistry , Troponin/metabolism , Troponin/ultrastructure
8.
Proc Natl Acad Sci U S A ; 102(14): 5038-43, 2005 Apr 05.
Article in English | MEDLINE | ID: mdl-15784741

ABSTRACT

Troponin senses Ca2+ to regulate contraction in striated muscle. Structures of skeletal muscle troponin composed of TnC (the sensor), TnI (the regulator), and TnT (the link to the muscle thin filament) have been determined. The structure of troponin in the Ca(2+)-activated state features a nearly twofold symmetrical assembly of TnI and TnT subunits penetrated asymmetrically by the dumbbell-shaped TnC subunit. Ca ions are thought to regulate contraction by controlling the presentation to and withdrawal of the TnI inhibitory segment from the thin filament. Here, we show that the rigid central helix of the sensor binds the inhibitory segment of TnI in the Ca(2+)-activated state. Comparison of crystal structures of troponin in the Ca(2+)-activated state at 3.0 angstroms resolution and in the Ca(2+)-free state at 7.0 angstroms resolution shows that the long framework helices of TnI and TnT, presumed to be a Ca(2+)-independent structural domain of troponin are unchanged. Loss of Ca ions causes the rigid central helix of the sensor to collapse and to release the inhibitory segment of TnI. The inhibitory segment of TnI changes conformation from an extended loop in the presence of Ca2+ to a short alpha-helix in its absence. We also show that Anapoe, a detergent molecule, increases the contractile force of muscle fibers and binds specifically, together with the TnI switch helix, in a hydrophobic pocket of TnC upon activation by Ca ions.


Subject(s)
Calcium/metabolism , Troponin C/chemistry , Troponin C/metabolism , Troponin T/chemistry , Troponin T/metabolism , Animals , Biophysical Phenomena , Biophysics , Chickens , Crystallography, X-Ray , Detergents , In Vitro Techniques , Models, Biological , Models, Molecular , Multiprotein Complexes , Muscle Contraction/physiology , Muscle, Skeletal/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
9.
J Biol Chem ; 279(22): 23504-9, 2004 May 28.
Article in English | MEDLINE | ID: mdl-14988396

ABSTRACT

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.


Subject(s)
Calmodulin-Binding Proteins/chemistry , Calmodulin/chemistry , Kinesins/chemistry , Plant Proteins/chemistry , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Binding Sites , Calmodulin/metabolism , Calmodulin-Binding Proteins/metabolism , Kinesins/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Solanum tuberosum/chemistry , Solanum tuberosum/metabolism
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