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 the production of interferon-g to initiate an antiviral response.


Figure 1. Dimeric Structure of Vaccinia virus VH1 determined crystallographically at 1.3Å resolution (PDB 3CM3). (A) Ribbon model of the dimeric phosphatase organization shown in two orientations. (B) Blow-up view of the active site showing VH1 catalytic triad and phosphate ion.

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 the 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.


Figure 2. Atomic structure of human DUSP26 determined at 1.68Å resolution (PDB 4HFR). (A) Ribbon diagram of DUSP26 crystallographic dimer (in side and top view) that is present in two copies in the asymmetric unit. Two protomers of a dimer (referred to as A and B) are colored in cyan and gray, respectively. (B) Blow-up view of DUSP26 active site reveals a trapped water molecule.

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 -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 -phosphate of RNA are defining features of atypical DSPs specialized in dephosphorylating 5'-RNA.


Figure 3. Atomic structure of PIR1-C152S-core determined at 1.20 Å resolution (PDB 4MBB and 4NYH). (A) Ribbon diagram of PIR1 phosphatase core. The active site phosphate and chloride ions are shown in red and yellow, respectively. (B) Magnified view of PIR1 final 2Fo-Fc electron density map calculated at 1.20 Å resolution and overlaid to the refined model of PIR1 P-loop (shown as sticks). 

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.


Figure 4. A structural model of human laforin derived using structural hybrid methods. (A) Ab initio SAXS reconstructions of dimeric laforin overlaid to ribbon models of laforin DSP (PDB 4R30) and CBM.(B) Agreement of P(r) functions calculated from experimental SAXS data (blue) and data calculated from the pseudo-model (red) of dimeric laforin. Inset panel. Experimental scattering data (blue) overlaid to the scattering curve calculated from the laforin model (red).