Structure of a Human A-Type Potassium Channel Interacting Protein DPPX, a Member of the Dipeptidyl Aminopeptidase Family

doi:10.1016/j.jmb.2004.09.003 J. Mol. Biol. (2004) 343, 1055–1065 Structure of a Human A-type Potassium Channel Interacting Protein DPPX, a Member of the Dipeptidyl Aminopeptidase Family Pavel Strop1, Alexander J. Bankovich2, Kirk C. Hansen3 K. Christopher Garcia2 and Axel T. Brunger1* 1Howard Hughes Medical It has recently been reported that dipeptidyl aminopeptidase X (DPPX) Institute and Departments of interacts with the voltage-gated potassium channel Kv4 and that Molecular and Cellular co-expression of DPPX together with Kv4 pore forming a-subunits, and Physiology, Neurology and potassium channel interacting proteins (KChIPs), reconstitutes properties Neurological Sciences, and of native A-type potassium channels in vitro. Here we report the X-ray Stanford Synchrotron Radiation crystal structure of the extracellular domain of human DPPX determined at Laboratory, Stanford University 3.0 A˚ resolution. This structure reveals the potential for a surface James H. Clark Center E300 electrostatic change based on the protonation state of histidine. Subtle 318 Campus Drive, Stanford changes in extracellular pH might modulate the interaction of DPPX with CA 94305, USA Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The 2Department of Microbiology dimeric structure of DPPX is highly homologous to the related protein and Immunology, Stanford DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of University School of Medicine the catalytic serine residue but the presence of an additional serine near the Fairchild D319, 299 Campus “active” site. However, the arrangement of residues is inconsistent with Drive, Stanford, CA 94305-5124 that of canonical serine proteases and DPPX is unlikely to function as a USA protease (dipeptidyl aminopeptidase). 3Department of Pharmaceutical q 2004 Elsevier Ltd. All rights reserved. Chemistry, University of California San Francisco San Francisco, CA, 94143-0446 USA Keywords: DPPX; DPP6; voltage gated potassium channel; dipeptidyl *Corresponding author aminopeptidase; serine protease Introduction proteins and peptides.9,10 DPP-IV, for example, plays an important role in regulation of physio- Dipeptidyl aminopeptidase X (DPPX, also logical processes that include immune, metabolic, termed DPP6, BSPL, or potassium channel accel- inflammatory, and central nervous system func- erating factor (KAF)) is a type II transmembrane tions11,12 by cleaving Xaa-Pro or Xaa-Ala from the protein belonging to the prolyl oligopeptidase N terminus of chemokines and neuropeptides. family of serine proteases.1–3 This family includes DPP-IV inactivates a variety of bioactive peptides proteins such as DPP-IV4 (CD26), fibroblast including glucagon hormones such as GLP-1, activation protein (FAP),5 prolyl oligopeptidase which stimulate insulin secretion. Therefore, (POP),6 DPP8,7 DPP9, and DPP10 (DPPY)8 DPP-IV is a potential therapeutic target for treating (K. Takimoto, personal communication). Prolyl type 2 diabetes. oligopeptidases remove dipeptides from regulatory Both DPPX and DPPY proteins have a substi- tution at the catalytic serine (aspartic acid in DPPX and glycine in DPPY) which appears to render them Abbreviations used: DPPX, dipeptidyl aminopeptidase 8,13 X; KChIP, potassium channel interacting protein; NCS, inactive proteases. Mutagenesis of the aspartic non-crystallographic symmetry; r.m.s.d., root-mean- acid residue in the active site back to serine in DPPX square deviation; IEF, isoelectric focusing. shows a twofold increase of protease activity E-mail address of the corresponding author: compared to the wild-type DPPX. However, this [email protected] DPPX variant engineered with a functional catalytic 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. C 1056 Structure of a K Channel Interacting Protein DPPX triad still has a 100-fold lower activity than wild- Results type DPP-IV.1,13 These data indicate that DPPX lacks the traditional proteolytic activity of the other Overall structure family members and that additional residues in the active site, apart from the catalytic triad, are DPPX exists as several splicing variants important for DPP-IV like activity.13 termed DPPX-L, DPPX-S, DPPX-N, and DPPX-O In the absence of a known proteolytic role, DPPX (K. Takimoto, personal communication). Here, we has been identified as an associated component of have chosen to use the numbering of the largest C an A-type K channel.3 The voltage-gated potass- variant (DPPX-L). In this variant, the intracellular N ium channel family (Kv) is a class of ion channels terminus (residues 1–95) is followed by a putative that play important roles in regulating the excit- transmembrane helix (residues 96–116) and a large ability of electrically active cells.14 The diversity of extracellular domain (residues 116–865). The extra- voltage-gated potassium channels is a result of both cellular domain crystallized in space group P21 a large number of Kv subunits, as well as with two dimers in the asymmetric unit. The interactions of Kv pore forming a-subunits with structure was determined by molecular replace- 12 auxiliary proteins.15,16 ment using DPP-IV as a search model, which has The Kv4 family of voltage-gated potassium 32% sequence identity and whose structure was ˚ channels is responsible for rapidly inactivating previously determined at 2.5 A. Six N-glycosylation 17 currents in the heart (ITO) and in neurons (ISA). sites were found in the DPPX structure with visible In the heart, Kv4 channels are responsible for initial electron density for at least two N-acetyl-glucosa- re-polarization of the cardiac action potential.18,19 In mine moieties. The first ten and last 16 residues of neurons, Kv4 channels regulate the propagation of the extracellular domain of DPPX were disordered. action potential and the firing frequency.20 In The final structure consists of 722 residues (127– dopaminergic neurons, firing frequency modulates 849) of the extracellular domain. Data, refinement, dopamine release and alterations in firing frequen- and model statistics are shown in Table 1. 21 The structure of DPPX closely resembles that of cies can induce Parkinsonian symptoms. Kv4 12,28–30 channels therefore provide therapeutic targets for the related DPP-IV. Two monomers associate diseases such as Parkinson’s and schizophrenia.22 to form a homodimer (Figure 1(a)). Each monomer b At present, Kv4 subunits are known to interact with consists of an eight-bladed -propeller domain (residues 142–322, and 351–581) (Figure 1(b)) and potassium channel interacting proteins (KChIPs) 3,23 an a/b hydrolase domain (residues 127–142, and and with DPPX/Y (K. Takimoto, personal 581–849) (Figure 1(c)). Both the a/b hydrolase communication). KChIPs are calcium-sensing domain and the b-propeller domain participate in proteins that increase the current amplitude and 15,24–26 the formation of the dimer, generating a buried alter Kv4 channel gating. DPPX associates surface area of 2574 A˚ 2 (9% of the total surface area a with Kv4 channels, facilitates Kv4 -subunits of a DPPX monomer). The dimer interface is formed trafficking to the membrane, and increases the rate 3,23 by the last b-strand and the last two helices in the of inactivation. DPPY was also found to a/b hydrolase domain and by the b-hairpin inser- modulate Kv4 channel properties in a similar, but tion motif from the b-propeller domain (Figure 1). In not identical fashion to DPPX27 (K. Takimoto, personal communication). DPPY also immuno- precipates with Kv4 subunits, increases Kv4 current density, and alters Kv4 channel gating Table 1. Refinement and model statistics 27 characteristics (K. Takimoto, personal communi- Space group P21 cation). Co-expression of DPPX, Kv4 pore forming Cell dimensions ˚ a-subunits, and KChIPs, reconstitutes properties of a, b, c (A) 69.0, 170.2, 159.3 3 b (8) 92.1 native A-type potassium channels in vitro. Resolution (A˚ ) 18.0–3.0 The binding site of DPPX on the Kv4 channel is Measured reflections 288,512 unknown at present. DPPX contains a very short Unique reflections 66,451 intracellular tail (31–95 residues depending on the Rmerge 9.8 (37.4) Completeness (%) 90.6 (63.3) isoform), one putative transmembrane helix, and a I/s(I) 15 (1.95) large 749 amino acid extracellular domain. The Number of: protein atoms 23,348 interaction between DPPX and Kv4 is most likely Carbohydrate atoms 825 mediated through the transmembrane helix and the Water molecules 51 Overall B-factor value (A˚ 2) 75.1 extracellular domain. Here we present the structure Rcryst (%) 24.8 of the extracellular domain of DPPX determined at Rfree (%) 28.0 3.0 A˚ resolution. Comparison of the active sites of Root-mean-square deviation DPPX and DPP-IV explains why DPPX is unable to Bonds 0.007 Angles 1.50 function as a dipeptidyl aminopeptidase. Based on Ramachandran the structure of DPPX and structural and bio- Most favored regions 72.5 chemical data for potassium channel interacting Additional allowed regions 24.0 proteins, we propose models of DPPX interactions Generously allowed regions 3.5 Disallowed regions 0.0 with Kv4 a-subunits. Structure of a KC Channel Interacting Protein DPPX 1057 Figure 1. Structure of DPPX. (a) Ribbon diagram of the DPPX dimer. The color changes from blue (N terminus) to red (C terminus). Carbohydrates with glycosylation sites are shown in ball-and-stick representation and the “catalytic triad” is shown in CPK representation. (b) View of the eight-bladed b-propeller domain (residues 142–322, and 352–581). (c) View of the a/b hydrolase domain (residues 127–142, and 581–849). The color scheme is the same as used in (a). The Figure was created with POVSCRIPT63,64 and POVRAY. solution DPPX exists primarily as homodimers; Active site however, small amounts of monomer, that over time self-associate to homodimers, were also The active site of the dipeptidyl peptidases is observed (data not shown).

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