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Dual Specificity Phosphatase 5‐Substrate Interaction: A Mechanistic Perspective

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ABSTRACT

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members’ abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5’s structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5’s mechanistic interaction and specificity toward phospho‐extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen‐activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449‐1461, 2017.

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Figure 1. Figure 1. Depiction of the active site of DUSP5 PD (A) as well as its juxtaposition with the X‐ray crystallography/NMR structures of DUSPs 3‐5, 8, 10, 13b, 14, 15, 18, 19, 22, and 27 () (B) showing their remarkable similarity in positioning of the catalytically relevant residues forming the active site (indicated by ellipses) as well as a histidine outside the reaction pocket (top right ellipse).
Figure 2. Figure 2. Chemical steps involved in dephosphorylation of the phosphorylated tyrosine of pY‐ERK2 by DUSP5.
Figure 3. Figure 3. Comparison of the X‐ray/NMR structures of DUSPs that are active toward ppERK1/2. Red ellipses show position of catalytic aspartic acid, blue ellipses highlight the conformational differences in the active site, and green‐, yellow‐, and pink‐filled ellipses show unique features associated only with DUSP5, i.e. glutamic acid E264, secondary binding site, and presence of a disulfide bridge, respectively ().
Figure 4. Figure 4. (A) Structure of the DUSP5/ppERK2 complex obtained by the computational modeling and molecular dynamics simulations. (B) Tertiary structure of EBD and ppERK2, where residues were colored according to their conservation score, that is, ranged from the cyan color corresponding to most variable residues to violet color corresponding to the most conserved ones (). Structures in the bottom of panel B show the frontal view of the interfacial regions of EBD and pERK2.
Figure 5. Figure 5. (A) The proposed sequence of binding events leading to formation of the DUSP5/ppERK2 complex. (B) Schematic representation of the binding of pERK and PD of DUSP5 leading to the prereactive near attack conformation. Yellow and black circles on the left show the position of active and secondary binding sites, respectively. Parts of panel B especially the schematic representation of the mechanism is modified from our previous publication ().


Figure 1. Depiction of the active site of DUSP5 PD (A) as well as its juxtaposition with the X‐ray crystallography/NMR structures of DUSPs 3‐5, 8, 10, 13b, 14, 15, 18, 19, 22, and 27 () (B) showing their remarkable similarity in positioning of the catalytically relevant residues forming the active site (indicated by ellipses) as well as a histidine outside the reaction pocket (top right ellipse).


Figure 2. Chemical steps involved in dephosphorylation of the phosphorylated tyrosine of pY‐ERK2 by DUSP5.


Figure 3. Comparison of the X‐ray/NMR structures of DUSPs that are active toward ppERK1/2. Red ellipses show position of catalytic aspartic acid, blue ellipses highlight the conformational differences in the active site, and green‐, yellow‐, and pink‐filled ellipses show unique features associated only with DUSP5, i.e. glutamic acid E264, secondary binding site, and presence of a disulfide bridge, respectively ().


Figure 4. (A) Structure of the DUSP5/ppERK2 complex obtained by the computational modeling and molecular dynamics simulations. (B) Tertiary structure of EBD and ppERK2, where residues were colored according to their conservation score, that is, ranged from the cyan color corresponding to most variable residues to violet color corresponding to the most conserved ones (). Structures in the bottom of panel B show the frontal view of the interfacial regions of EBD and pERK2.


Figure 5. (A) The proposed sequence of binding events leading to formation of the DUSP5/ppERK2 complex. (B) Schematic representation of the binding of pERK and PD of DUSP5 leading to the prereactive near attack conformation. Yellow and black circles on the left show the position of active and secondary binding sites, respectively. Parts of panel B especially the schematic representation of the mechanism is modified from our previous publication ().
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Teaching Material

R. G. Kutty, M. R. Talipov, R. D. Bongard, R. A. J. Lipinski, N. L. Sweeney, D. S. Sem, R. Rathore, R. Ramchandran. Dual Specificity Phosphatase 5-Substrate Interaction: A Mechanistic Perspective. Compr Physiol 7: 2017, 1449-1461. doi:10.1002/cphy.c170007

Didactic Synopsis

  1. Understanding the structural features in proteins is key to identifying strategies for pursuing them as drug targets. Dual specific phosphatase 5 (DUSP5) contains two binding pockets, both of which are responsible for interaction with phosphate residues in its substrate ERK. Entry of the threonine phosphate residue in ERK into the secondary pocket (secondary site) facilitates accessibility for the tyrosine phosphate residue in ERK to the primary pocket (active site) of DUSP5. This is dubbed the “lock-and-key,” mechanism with the “key” being entry of threonine phosphate residue into secondary pocket, which then “unlocks” the active site for the tyrosine phosphate binding.
  2. Phosphatases as drug targets are not developed to the same extent as their counterpart kinases. The structural similarities between all members of the DUSP family underscore this point. DUSP5 compared to other DUSPs shares unique structural features that help explain its specificity to substrate ERK. The unique mechanism described in teaching point 1 allows for specific targeting of the DUSP5:ERK interface.
  3. Numerous skillsets and tools are necessary for successful drug discovery projects. This includes skillsets from various scientific disciplines, including biochemistry, organic chemistry, and target biology. Tools include molecular dynamics simulation, molecular docking, enzymology, and organic synthesis. Complementary expertise across disciplines with expertise in specific tools is often necessary to solve complex problems in drug discovery.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1. Teaching points: The catalytic site is very highly conserved between DUSPs, both inside and outside of the binding pocket. This high degree of homology underscores the importance of the active site and suggests a common mechanism for dephosphorylation between DUSPs.

Figure 2. Teaching points: The aspartic acid residue (Asp232) of DUSP5 serves as both acid and base in the dephosphorylation of ERK by DUSP5. First, it serves as general acid and provides a proton to the leaving tyrosine/threonine. Second, it acts as a general base in the addition of the hydroxyl group from water to the phosphorylated cysteine.

Figure 3. Teaching points: DUSPs show very highly conserved sequence homology. However, this sequence homology does not always lend itself to similarity in tertiary structure. Specifically, the location of the critical aspartic acid residue discussed in Figure 2 is not spatially conserved. This residue may be near or far from the pocket, depending on the DUSP species in question. Additionally, there are several key unique features of DUSP5—presence of a disulfide bridge (pink ellipse), a secondary binding site (yellow ellipse), and a glutamic acid residue (green ellipse).

Figure 4. Teaching points: Full-length DUSP5 has not been crystalized and therefore, no full X-ray crystallographic structure is available. However, based on the high sequence homology between DUSP5 and other DUSPs it is possible to render a very close approximation of the structure of DUSP5. High confidence in these models is enforced through conservation comparisons (B). In this model, the most conserved residues of the ERK Binding Domain (EBD) should be on the face closest to the ERK molecule itself, which is the case. These models demonstrate the ability to infer structures of unsolved proteins through homology and molecular simulations with a high degree of confidence.

Figure 5. Teaching points: The mechanism of precatalysis arrangement of the DUSP5:ERK2 protein complex is a concerted, multistep process. A shows the domain version of the DUSP5 and ERK2 proteins while B shows the catalysis reaction in detail as it relates to active and secondary sites of phosphatase domain (PD) of DUSP5 with TEY motif of ERK2 protein. The steps in the reaction for panel A are: (1) The EBD interacts with the C-terminus of ERK, aligning the N-terminus of the DUSP5 linker (orange wavy line) to a groove in ERK (yellow region). (2) This alignment forces a zipper-like arrangement of the linker into the ERK groove. (3) The pT and pY residues (black dots on red interface) are brought into the proximity of the phosphatase domain. (4) The close proximity of the EBD and PD domain of DUSP5 enveloped around ppERK2 is shown. The steps in the reaction for panel B are: (1) The active site (yellow circle) and the secondary site (black circle) in the phosphatase domain of DUSP5 are depicted in the ribbon structure format, and are also shown in a simplified cartoon format. (2) The pT residue of ERK2 enters the secondary pocket first, interrupting the salt bridge (red vertical rectangles) formed between R269 and E264. (3) The pY residue of ERK2 is then able to enter the active site, forming the prereactive complex.


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Raman G. Kutty, Marat R. Talipov, Robert D. Bongard, Rachel A. Jones Lipinski, Noreena L. Sweeney, Daniel S. Sem, Rajendra Rathore, Ramani Ramchandran. Dual Specificity Phosphatase 5‐Substrate Interaction: A Mechanistic Perspective. Compr Physiol 2017, 7: 1449-1461. doi: 10.1002/cphy.c170007