Cingolani Research

Contact

Name: Gino Cingolani, PhD
Position: Professor

233 South 10th Street
BLSB 826
Philadelphia, PA 19107

Telephone: 215-503-4573

My lab uses biophysical, biochemical and cellular techniques to study the structure and function of biological macromolecules. We employ rigorous chemical and physical methodologies to solve medically-relevant problems that help to decipher the most fundamental mechanisms of life and contribute to improving human health.

General areas of research include protein nuclear import, viral genome ejection/packaging motors, multi-subunits ATPases and disease-linked phosphatases.

We are located at the 8th floor of the Bluemle Life Sciences Building, in the Department of Biochemistry and Molecular Biology at Thomas Jefferson University, downtown Philadelphia.

Research Projects

Nucleocytoplasmic Transport

Nucleocytoplasmic transport is central to the functioning of eukaryotic cells and is an integral part of the processes that lead to most human diseases. Over the past fifteen years, we have elucidated the molecular basis for recognition of classical (Cingolani et al., Nature, 1999) and non-classical (Cingolani et al., Mol. Cell, 2002) import substrates by the ubiquitous transport factor importin b (also known as Karyopherin b). In the so called 'classical' import reaction, the receptor importin b heterodimerizes with the adaptor importin a, that in turn associates directly with proteins bearing a classical Nuclear Localization Signal (NLS) via its helical Arm-core (Fig. 1). The trimeric import complex docks to the Nuclear Pore Complex (NPC) and translocates through the NPC via multiple rounds of interactions of importin b with FG-rich nucleoporins lining the NPC, in a process that requires the GTPase Ran.

The human genome encodes seven isoforms of the adaptor importin a that share high sequence similarity (Pumroy and Cingolani, Biochemical J., 2015) (Fig. 2). All isoforms share a fundamentally conserved architecture that consists of an N-terminal auto-inhibitory importin b binding (IBB)-domain and a C-terminal Arm-core that associates with NLS-cargos. Despite striking similarity in aminoacid sequence and 3D-structure, importin α isoforms display remarkable substrate specificity in vivo. While all importin a isoforms can import substrates bearing a classical NLS, only certain isoforms bind to dimeric transcription factors like STATs and NF-kB(p50:p65), a phenomenon that we named 'isoform-specialization'.

In my laboratory, we are interested in understanding the mechanisms by which nuclear transport is regulated under physiological conditions and in diseased states. Our research is important to understand how living cells regulate the availability of essential factors bearing nuclear activity (e.g. transcription factors, DNA replication factors, etc). This is emerging as a novel and very powerful way to control gene expression and cellular differentiation. Likewise, misregulation of nuclear transport both in over-proliferating tumor cells and in cells hijacked by pathogenic viruses, is emerging as a critically important target for pharmacological intervention.

Viral Genome Delivery

The goal of our research is to understand how double stranded DNA viruses deliver their large genomes (~40-250kb) into living cells. Most of the work carried out in my laboratory is based on a simple model system, the Salmonella-phage P22. Using a combination of structural (e.g. X-ray crystallography, electron cryo-EM, SAXS), biochemical and functional techniques, we investigate the structure, composition and assembly of P22 genome ejection machinery. In collaboration with Dr. Sherwood Casjens (University of Utah) we have isolated and reconstituted the five polypeptide chains forming P22 tail machinery and determined their assembly and structural composition in vitro. In collaboration we Dr. Jack Johnson (at the Scripps Research Institute) and Dr. Timothy Baker (UCSD), we determined a 7.8A asymmetric cryo-EM reconstruction of P22 mature virion (Fig. 1A) (Tang et al., Structure, 2011). This wonderful structure served as molecular framework to identify and fit individual components previously solved in my laboratory, namely the dodecameric portal protein (Fig. 1B), gp4 (Fig. 1C) (Olia et al., Nature Struc Mol Biol., 2011) and the tail needle gp26 (Fig. 1D) (Olia et al., Nature Struc Mol Biol., 2007). Overall, the synergy of top-down (e.g. cryo-EM) and bottom-up approaches (e.g. high resolution crystal structures of individual components) provides an invaluable tool to study the mechanisms of viral genome ejection at the most fundamental molecular level.

A second project in the lab focuses on viral genome packaging, which is the mirror process of genome ejection. Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. We recently described the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation (Fig. 2) (Roy et al., Structure, 2012). The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology.

We also characterized the structure and regulation of P22 L-terminase subunit (gp2) (Roy and Cingolani, J. Biol Chem, 2012). This protein has a bipartite organization, consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in drop proteolysis of full length L-terminase reveals a central seven-stranded b-sheet core that harbors two magnesium ions (Fig. 3). Modeling studies with DNA suggest the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA-binding surface is sterically hindered by a loop-helix motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, while it exhibits endonucleolytic activity in the context of FL-L-terminase. Deleting the auto-inhibitory L1-a2 motif (or just the loop L1) restores nuclease activity to a level comparable to FL-L-terminase. Together, these results suggest that the activity of P22 headful nuclease is regulated by an intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome-packaging.

Disease-linked Phosphatases

Protein Tyrosine Phosphatases (PTPs) are essential signaling enzymes critically linked to human diseases. PTPs are broadly grouped into four subfamilies, of which the first and largest subfamily, Class I-PTPs, consists of classical PTPs, which dephosphorylate exclusively phospho-Tyr and Dual Specificity Phosphatases (DSPs) that can hydrolyze phospho-Tyr and phospho-Ser/Thr. Since the discovery of the first DSP, VH1, encoded by Vaccinia virus, 61 VH1-like DSPs have been identified in all kingdoms of life. The human genome encodes 38 VH1-like DSPs (also referred to as 'DUSPs') that function as critical signaling molecules, central to cell physiology and involved in a myriad of pathological processes that lead to disease. Current work in my laboratory is aimed at understanding the structure, substrate-specificity and regulation of disease-associated DSPs. 

We have determined the crystal structure of the Vaccinia virus VH1 at 1.3Å resolution (Koksal et al., J. Biol Chem, 2009). VH1 adopts a novel dimeric quaternary structure, stabilized by an N-terminal domain swap (Fig. 1) that exposes two active sites spaced ~39Å away from each other. Two to three hundred copies of VH1 are encapsidated in a Vaccinia virion and released in the infected cell upon virus entry. We demonstrated that in the cytoplasm of infected cells, VH1 specifically dephosphorylates the transcription factor STAT1 at Tyr701 (Koksal and Cingolani, J. Biol Chem, 2011), blocking its nuclear translocation. Retention of STAT1 in the cytoplasm prevents production of interferon-g to initiate antiviral response.

DUSP26 is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-Ser20 and phospho-Ser37 in the p53 transactivation domain (TAD). We recently reported the 1.68Å crystal structure of a catalytically inactive mutant (Cys152Ser) of DUSP26 lacking the first N-terminal 60 residues (Fig. 2A). Our structural analysis (Lokareddy et al., Biochemistry, 2013) revealed that DUSP26 adopts a closed conformation of the protein tyrosine phosphatase (PTP)-binding loop, which results in an unusually shallow active site pocket and buried catalytic cysteine. A water molecule trapped inside the PTP-binding loop makes close contacts both with main chain and side chain atoms (Fig. 2B). As in the case of the phosphatase MKP-4, a substrate-induced conformational change, possibly involving rearrangement of helix a9 with respect to the phosphatase core, may allow DUSP26 to adopt a catalytically active conformation. Since regulation of p53 phosphorylation is critical to control its stability and biological activity, inhibition of DUSP26 is a potential target to enhance p53-mediated response, which could be useful to treat neuroblastomas insensitive to chemotherapy and increase the success of treatment. The high resolution crystal structure of DUSP26 provides the first atomic insight into this disease-associated phosphatase.

PIR1 is an atypical dual specificity phosphatase (DSP) that dephosphorylates RNA with higher specificity than phosphoproteins. We have solved the atomic structure of a catalytically inactive mutant (C152S) of human PIR1 phosphatase core, refined at 1.20 Å resolution (Sankhala et al.Biochemistry, 2014) (Fig. 3A). PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5'-phosphatases such as the Baculovirus RNA Triphosphatase (BPV) and the human mRNA capping enzyme. PIR1 active site cleft is wider and deeper than in VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the g-phosphate of RNA and, ~6 Å away, a chloride ion coordinates the general base R158. Two residues in PIR1 phosphate-binding loop (P-loop), a histidine (H154) downstream of C152 and an asparagine (N157) preceding R158, make close contacts with the active site phosphate and their non-aliphatic side chains are essential for phosphatase activity in vitro. These residues are conserved in all RNA 5'-phosphatases that, analogous to PIR1, lack a 'general acid' residue. Thus, a deep active site crevice, two active site ions and conserved P-loop residues stabilizing the g-phosphate of RNA are defining features of atypical DSPs specialized in dephosphorylating 5'-RNA.

Laforin is another important disease-linked phosphatase studied in my laboratory. Encoded by the EPM2A gene, laforin is mutated in patients suffering from Lafora disease, a fatal form of progressive myoclonic epilepsy. Laforin removes phosphate groups from glycogen during biosynthetic activity. Loss of function mutations in the gene encoding laforin is the predominant cause of Lafora disease (LD), a fatal form of progressive myoclonic epilepsy. We used hybrid structural methods to derive a complete structural model of human laforin (Sankhala et al.J Biol Chem, 2015). We found that laforin adopts a dimeric quaternary structure, topologically similar to VH1 (Fig. 4). The interface between laforin carbohydrate-binding module (CBM) and DSP domain generates an intimate substrate-binding crevice that allows for recognition and dephosphorylation of phosphomonoesters of glucose. We identify novel molecular determinants in laforin active site that help decipher the mechanism of glucan phosphatase activity.

Multi-subunits ATPases

ATP synthases form an evolutionarily related family of energy-coupling, ion-transporting enzymes that is responsible for the synthesis of most cellular ATP in plants, animals, and many bacteria. ATP synthases function as dual-engine rotary motors. A membrane-embedded complex (F0) acts as a turbine to transport ions (protons, but Na+ in some bacteria). A peripheral stator stalk and a central rotor stalk connect F0 to an extrinsic complex (F1) in which rotation of the asymmetric central stalk coordinates ATP synthesis or hydrolysis.

For over 30 years, the ATP synthase of the gram negative bacterium Escherichia coli (EcF1) has provided the predominant system for classical genetics and mutagenesis studies of the functional mechanism of ATP synthases. Bacterial ATPases have also been recognized as a novel and powerful target for the development of anti-bacterial drugs. A new class of effective anti-tuberculosis drugs that kills mycobacteria specifically inhibits the F0 subunit (Andries et al, 2005; Koul et al, 2007). Despite the growing importance of this multi-subunit enzyme in biology and medicine, the 3D-structure of the prototypical EcF1 enzyme has remained elusive. EcF1, a multi-protein complex formed by 9 proteins (α3, β3, γ, ε, δ), is in fact highly resistant to crystallization and has always failed to yield high quality crystals despite the effort of many laboratories worldwide.

In collaboration with Dr. Thomas Duncan, at SUNY Upstate Medical University, we have identified a stable and enzymatically active 8-protein catalytic core of EcF1 (EcF1-δ) (Fig. 1A). We crystallized EcF1-δ using a combination of robotic and manual crystallization techniques (Fig. 1B, C) and were able to collect complete diffraction data at NSLS beamline X25 and X6A. EcF1-δ crystals belong to space group C2 with unit cell parameters a=435.97Å, b=183.06Å, c=225.39Å and β-angle = 108.99°; the asymmetric unit contains four EcF1-δ complexes (~18,500 residues!). The 3D-structure of EcF1-δ was solved at 3.2Å resolution and refined to an Rwork/Free ~24.3/26.4%. This work was published in the 2011 June issue of Nature Structural and Molecular Biology (Cingolani & Duncan, 2011). The general architecture of EcF1 is analogous to that of mitochondrial F1 (Abrahams et al, 1994) and is illustrated in Fig. 1D,E. The structure contains an hexameric ‘catalytic ring’ of α- and β-subunits surrounding the upper region of a ‘central stalk’, which consists of γ and ε-subunits. Nucleotide binding sites on β subunits are responsible for ATP synthesis/hydrolysis, while sites on α subunits are noncatalytic.

Perhaps the most striking and novel feature of the EcF1 structure lies in the C-terminal domain of subunit ε (ε CTD), that adopts a highly extended conformation, deeply inserted into the central cavity of the enzyme (Fig. 1D). This extended conformation of the ε-subunit contrasts with ε’s homolog in MtF1 structures (Gibbons et al, 2000) and with the compact state observed for isolated E. coli ε-subunit (Uhlin et al, 1997; Wilkens & Capaldi, 1998; Yagi et al, 2007) (Fig. 2B). The extended conformation of ε-CTD in EcF1 engages both γ-subunit and catalytic ring in extensive contacts that are incompatible with functional rotation. While the extended conformation of εCTD visualized crystallographically agrees with previous chemical labeling and cross-linking studies (Wilkens & Capaldi, 1998), this structure provides a rational explanation for the inhibitory role of ε-subunit, which is well documented for ATP synthases of bacteria and chloroplasts. Regulation of ATP synthase activity by ε-subunit is however not present in the mitochondrial enzyme. As functional ATP synthase is critical for the viability of pathogenic bacteria such as Streptococcus pneumonia, Mycobacterium tuberculosis, differences in structural complexity and regulation between bacterial and mitochondrial ATP synthases can be exploited to selectively inhibit pathogenic bacteria. Thus, the structure of the autoinhibited EcF1 provides the structural basis to understand ε-subunit mediated inhibition of rotary catalysis and gives us a rational framework for developing antimicrobial agents that selectively mimic or stabilize the ε-inhibited state of bacterial ATP synthases but do not inhibit mitochondrial ATP synthases.

In the future, we will continue the structural characterization of the bacterial ATPases with the following specific aims:

  • To determine the structure of the EcF1 in the uninhibited conformation
  • To visualize the conformation of ε-subunit in the Ec-F0F1 holoenzyme.
  • To map structurally the conformation of ε-subunit in F1 ATPases of pathogenic bacteria.