Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405001200 on June 22, 2004

J. Biol. Chem., Vol. 279, Issue 41, 43330-43335, October 8, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/41/43330    most recent
M405001200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weihofen, W. A.
Right arrow Articles by Fan, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weihofen, W. A.
Right arrow Articles by Fan, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Crystal Structure of CD26/Dipeptidyl-peptidase IV in Complex with Adenosine Deaminase Reveals a Highly Amphiphilic Interface*

Wilhelm A. Weihofen{ddagger}, Jiango Liu§, Werner Reutter§, Wolfram Saenger{ddagger}, and Hua Fan§||

From the {ddagger}Institut für Chemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany and the §Institut für Molekularbiologie und Biochemie, Charité-Universitätsmedizin Berlin, Arnimallee 22, D-14195 Berlin, Germany

Received for publication, May 5, 2004 , and in revised form, June 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a {beta}-propeller domain and an {alpha}/{beta}-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-Å resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each {beta}-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the {beta}-propeller domain of DPPIV interact with two hydrophilic and heavily charged {alpha}-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
CD26 or dipeptidyl-peptidase IV (DPPIV,1 EC 3.4.14.5 [EC] ) is a ubiquitous, multifunctional integral type II membrane glycoprotein located on the surface of a variety of epithelial, endothelial, and lymphoid cells, and it also occurs in soluble form in serum. As serine exopeptidase, DPPIV cleaves N-terminal dipeptides from polypeptides with proline or alanine preferentially in the penultimate position, thereby regulating the activity of a variety of biologically important peptides (1). Besides its peptidase activity, one of the main functions of DPPIV is binding to adenosine deaminase (ADA, EC 3.5.4.4 [EC] ) and to the extracellular matrix (2). All of these functions can influence T-cell proliferation (3, 4). In addition, DPPIV interacts with human immunodeficiency virus-1 Tat protein (5), and the human immunodeficiency virus envelope protein gp120 inhibits binding of DPPIV to ADA (6).

Crystal structures of DPPIV free and in complex with different inhibitors show that DPPIV is a "U" -shaped homodimer, the two monomers being related by a pseudo-2-fold rotation axis coinciding with the symmetry axis of the "U" (3, 7-9). The six-residue-long N-terminal cytoplasmic tails of the DPPIV dimer are followed by 22-residue-long transmembrane {alpha}-helices. The subsequent extracellular ectodomains are divided into two domains each. Eight-bladed {beta}-propeller domains (Arg54-Asn497) form the arms of the "U" distal to the membrane plane, and {alpha}/{beta}-hydrolase domains (Gln508-Pro766) proximal to the membrane form the bend of the "U" and harbor the catalytic triads that are required for peptidase activity.

Soluble DPPIV (without transmembrane {alpha}-helix) migrates as a dimer in gel filtration (3) but can also form higher molecular weight assemblies with apparent mass of 900 kDa (10). This could be due to a tetrameric assembly as observed in the crystal structure of porcine DPPIV (pDPPIV) that is associated with head-to-head binding between the ends of the arms of two "U" -shaped DPPIV dimers to form a " " -shaped complex. Engel et al. (9) suggested that pDPPIV tetramerization might be involved in cell-cell contacts, and this view would explain why DPPIV promotes adhesion between lymphocytes and epithelial cells that is inhibited by the addition of exogenous ADA (11).

ADA is a ubiquitous, soluble, and globular enzyme with a TIM barrel fold (eight parallel {beta}-strands forming a barrel decorated by {alpha}-helices) (12). It is present in all mammalian tissues and involved in the development and function of lymphoid tissue. ADA binds specifically to the DPPIV of humans, cattle, and rabbits with dissociation constants of 3 to 20 nM depending on the organism (3). Binding of ADA to DPPIV is important in regulating the extracellular local concentration of adenosine (13, 14). Inhibition of ADA reduces signals mediated by CD3 and T-cell receptors and suggests a correlation between ADA binding to DPPIV and T-cell activation (15), as elevated adenosine concentration inhibits the proliferation of T-lymphocytes. Although they share high sequence homology with their higher mammal analogs, DPPIV and ADA of rodents do not form a complex.

A cryo-electron microscopy study of the DPPIV·ADA complex at 22 Å resolution confirmed the location of the ADA binding site on the {beta}-propeller domain of DPPIV (16). Because details of intermolecular interactions remained elusive at this low resolution, ADA was oriented relative to DPPIV on the basis of mutagenesis studies, which suggested that helix {alpha}2 of ADA binds to segments of {beta}-propeller blades IV and V of DPPIV (17-19).

We crystallized the complex of human DPPIV (hDPPIV) ectodomain with bovine ADA (bADA), which shares 91% amino acid sequence homology with human ADA (hADA). The crystal structure determined at 3.0 Å resolution shows the intermolecular contacts in a highly amphiphilic interface that contribute to and stabilize hDPPIV·bADA complex formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Production, Purification, and Crystallization of hDPPIV·bADA—Full-length, enzymatically active hDPPIV was expressed in Sf9 cells and purified (20). N-terminal sequencing showed that residues 1-29 were uniformly truncated during purification. The hDPPIV·bADA complex was prepared by mixing hDPPIV and bADA (Sigma type VIII from calf intestinal mucosa) with a molar excess of bADA in 20 mM Tris, 500 mM NaCl, pH 8.0, and incubating at 4 °C overnight. The formed complex was analyzed by gel electrophoresis under nondenaturing conditions (20), purified by gel filtration on Superdex 200 10/30 (Amersham Biosciences) with 20 mM Tris-HCl, 150 mM NaCl, pH 8.0 (16), and concentrated to 7 mg/ml. For crystallization, a reduced factorial screen (Hampton Research) was set up using the vapor diffusion method. Crystals were obtained with 20-22% polyethylene glycol 3350, 200 mM NaCl, and 100 mM Tris-HCl, pH 8.0, and optimized by microseeding. Prior to data collection, the crystals were soaked in 25% polyethylene glycol 3350, 200 mM NaCl, 100 mM Tris, pH 8.0, supplemented with 20% glycerol, and cryo-cooled. Diffraction quality improved by annealing the crystal to room temperature twice for 3 s.

Structure Determination—X-ray data were collected at BESSY II, Berlin, Germany (Beamline BL1 of Free University, Berlin) and processed with DENZO and SCALEPACK (21); see Table I. The structure of hDPPIV·bADA was solved by a molecular replacement method with the program MOLREP (22) using glycoside-depleted hDPPIV as a partial search model (Protein Data Bank (PDB) code 1N1M [PDB] ), yielding two DPPIV dimers per asymmetric unit. Difference electron density clearly showed the presence of ADA molecules. Although an additional search using bADA (PDB code 1KRM [PDB] ) located four bADA molecules, the asymmetric unit is occupied by two (hDPPIV·bADA)2 complexes. Refinement was done using the REFMAC5 program (23) by applying four-fold noncrystallographic symmetry restraints to the protein chains but omitting the interface regions (24). Manual model building was done with the program O (25). Glycoside residues could be fitted to the electron density at eight of the expected nine N-glycosylation sites except for Asn685. For statistics, see Table I. The figures of molecules were prepared with MolScript (26) and RASTER3D (27).


View this table:
[in this window]
[in a new window]
  		TABLE I
Crystallographic data and refinement statistics

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure—hDPPIV was expressed and purified as full-length protein, but N-terminal sequencing revealed that it was cleaved during purification after residue 29, explaining its solubility in detergent-free buffer. The final electron density map of hDPPIV·bADA featured no density for the N-terminal residues 30-38 presumably because of high flexibility and/or disorder of this segment. Ser39 is the first residue with appropriate electron density of the hDPPIV ectodomain.

The crystal asymmetric unit contains two complexes, each with (hDPPIV·bADA)2 stoichiometry and a molar mass of 275 kDa (Fig. 1A). Each hDPPIV binds one bADA at the periphery of the {beta}-propeller domain near the ends of the arms of the "U" (Fig. 1B), thereby preserving the pseudo-two-fold symmetry of the complex (Fig. 1A). All crystal contacts of (hDPPIV:bADA)2 are distant to the interface region, suggesting that they will not structurally influence the binding of hDPPIV to bADA. Superposition of main chain structures of hDPPIV (PDB code 1N1M [PDB] ) and bADA (PDB code 1KRM [PDB] ) with their equivalents in (hDPPIV·bADA)2 yielded average root mean square deviations of 0.8 and 0.9 Å, respectively, indicating that their structures are nearly identical. In particular, the interface regions of hDPPIV and bADA do not rearrange significantly upon complex formation and show root mean square deviations of 1.0 and 0.6 Å, respectively, for main chain atoms. In the (hDPPIV·bADA)2 complex, bADA does not block the possible path of substrate through the central tunnel of the {beta}-propeller or through the side opening to the active site of hDPPIV (Fig. 1), and conversely, binding of hDPPIV (Fig. 1B) does not block the active site of bADA. This agrees with studies indicating that upon complex formation, DPPIV and ADA remain catalytically active (28).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 1.
Overall structure ("U") of the (hDPPIV·bADA)2 complex. A, the membrane and membrane anchor (not seen in the electron density) are drawn schematically. The view is normal to the pseudo-two-fold axis (vertical arrow) that relates the two hDPPIV·bADA units in (hDPPIV·bADA)2. The domains of hDPPIV are violet and blue for the {alpha}/{beta} hydrolase and {beta}-propeller domains, respectively; bADA is shown in green, active site Zn2+ is shown as a red sphere, and the oligosaccharide at conserved DPPIV-Asn229 is shown in stick representation. Access arrow 1 points to the entrance of the substrate to the active site through the {beta}-propeller, and arrow 2 points through the side opening of hDPPIV. Orientations of the axes of the TIM barrels of bADA are represented by black lines. In the hDPPIV·bADA binding sites, loops A and B of hDPPIV are red, and {alpha}-helices {alpha}1 and {alpha}2 of bADA are yellow. B, view along the axis of the propeller domain of DPPIV, rotated by ~40° around the horizontal so that bADA moves toward the viewer. The violet {beta}-strand N-terminal of loop A (red) is engaged in the tetramer formation of porcine DPPIV. The asterisk indicates the position of glycosylated Asn281.

 
The hDPPIV·bADA Interface—The hDPPIV:bADA interface buries 930-Å2 solvent-accessible surface area/polypeptide corresponding to 3 and 7% of the total accessible surface of individual hDPPIV and bADA molecules, respectively. Four glycoside residues linked to the conserved Asn229 of hDPPIV are well defined in the electron density (Fig. 2). They contact 33Arg-Arg-Gly-Ile36 of bADA and form one intermolecular hydrogen bond, and the protein-sugar interactions increase the buried surface by 170 Å2. This contact, however, is not a prerequisite for ADA binding as shown by site-directed mutagenesis of Asn229 -> Ala or deletion of any other N-glycosylation site (29).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 2.
Glycoside residues bound to Asn229 of hDPPIV interact with 33Arg-Arg-Gly-Ile36 of bADA. Electron density is drawn at the 1.2 {sigma} level. Carbon atoms are gray, oxygen is red, and nitrogen is blue. The dashed black line indicates a hydrogen bond (Man3O2H:Gly35O, 2.6 Å). MAN, mannose; NAG, N-acetylglucosamine.

 
hDPPIV binds bADA with two adjacent loops. Loop A (Ile287-Asp297) connects blades IV and V, and loop B (Asp331-Gln344) links {beta}-strands {beta}3 and {beta}4 of blade V (Fig. 3). The loops protrude from the propeller blades and form a cleft accommodating helix {alpha}2 (Pro126-Asp143) of bADA (Fig. 4A). Interactions between helix {alpha}1 (Arg76-Ala91) of bADA and loop A of hDPPIV complete the interface (Fig. 4). As indicated in Fig. 3 by triangles, 14 and 13 residues in hDPPIV and bADA, respectively, are engaged in intermolecular contacts at the binding interface (see Fig. 4B). The supplementary information provides all interfacial contacts shorter than 3.9 Å.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3.
Amino acid sequence alignment of DPPIV and ADA binding segments. Black triangles indicate amino acids engaged in complex formation. Identical and different amino acids are shown with gray and white background, respectively. Altered positions are in italics, and letters in bold italic denote nonconserved residues in rodents (R. norvegicus, M. musculus). Top, segment of human, rat, and mouse DPPIV with {beta}-strands indicated by arrows labeled with {beta}-propeller blades in roman and {beta}-strands in arabic numbers, respectively. Loop A and loop B indicate segments of DPPIV engaged in hDPPIV·bADA complex formation and the tetramer segment is engaged in tetramer formation in porcine DPPIV. Bottom, segment of human, bovine, and rodent (rat and mouse) ADA binding to DPPIV. {alpha}-Helices are indicated and labeled as {alpha}1 and {alpha}2.

 



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4.
The interface between bADA and hDPPIV. A, stereo plot of the hDPPIV·bADA interface, with bADA segments in green and hDPPIV segments in gray. Hydrogen bonds are indicated by black dashed lines. B, schematic representation of interactions between hDPPIV and bADA residues. Carbon atoms are shown in black, and nitrogen and oxygen atoms are as described in the legend to Fig. 2. Distances are in Å. Direct hydrogen bonds between proteins are shown as green dashed lines, and hydrophobic contacts are indicated by red eyelashes. This figure was partly generated with Ligplot (34).

 
A total of 11 intermolecular hydrogen bonds are formed between hDPPIV and bADA (Fig. 4). bADA-Glu139 on helix {alpha}2 appears to be important, as the carboxylate oxygen O{epsilon}1 forms hydrogen bonds to loop A of hDPPIV involving hDPPIVAla291NH, hDPPIV-Ser292NH, and hDPPIV-Ser292O{gamma}. In addition, bADA-Glu139O{epsilon}2 is hydrogen-bonded to hDPPIV-Gln344N{epsilon} on loop B, and hDPPIV-Gln344O{epsilon} is in contact with bADA-Arg142N{eta} on helix {alpha}2. The array of 11 hydrogen bonds with one hydrogen bond per 169 Å2 of buried surface is in close agreement to the average value of 10 hydrogen bonds per protein-protein interface and one hydrogen bond per 170 Å2 of buried interface area (30).

The interface in the hDPPIV·bADA complex is strongly amphiphilic. Of the 14 residues of hDPPIV forming intermolecular contacts at the interface, seven are apolar, six neutral polar, and only one (Arg336) is charged (Figs. 3, 4B, and 5). This agrees grossly with the average distribution of residues in protein-protein interfaces, which comprise 57% apolar, 24% neutral polar, and 19% charged residues (30). By contrast, all of the 13 residues forming the interface region of bADA are polar, and among these, 9 (69%) are charged (see Fig. 5). The charged residues of bADA form two intramolecular and one intermolecular salt bridges (bADA-Arg76···bADA-Glu128, bADA·Arg142···bADA-Asp143), and (bADA-Asp127···hDPPIVArg336, respectively), and the other charged residues of bADA form hydrogen bonds to hDPPIV (Fig. 4). The highly charged interface region of bADA is unusual. Of all 27 residues participating in complex formation at the hDPPIV·bADA interface, 10 (37%) are charged, which is the highest percentage ever observed in protein·protein complexes, with the next highest value being 27% (30). As the sequences of human and bovine ADA are identical at the binding interface (Fig. 3), except for the conservative substitution hADA-Glu77/bADA-Asp77 that forms a hydrogen bond to hDPPIV-Gln286N{epsilon} (Fig. 4), structures of hDPPIV in complex with bovine and human ADA are supposed to be nearly identical.



View larger version (66K):
[in this window]
[in a new window]
  		FIG. 5.
Surface representation of the hDPPIV·bADA interface. This figure was drawn with GRASP (35). Contacting residues (distance cutoff, 3.9 Å) are assigned in one-letter code followed by sequence number. Positively and negatively charged residues are blue and red, unpolar and polar but uncharged residues are yellow and orange, respectively. The bold arrow indicates the access to the active site through the {beta}-propeller opening of DPPIV and the thin curved double arrow indicates the salt bridge between hDDPIV-Arg336 and bADA-Asp127. The two parallel arrows indicate the location and orientation of {alpha}-helices {alpha}1 and {alpha}2 of bADA as depicted in Figs. 1 and 3.

 
Binding Studies Are Consistent with the (hDPPIV·bADA)2 Structure—ADA from mouse and rat does not bind to endogenous DPPIV but binds weakly (in the µM range) to the DPPIV of primates, cattle, and rabbits. Although this lack of complex formation in rodents might be associated with amino acid substitutions in loops A and B of DPPIV and in helix {alpha}2 of ADA (Fig. 3), it has inspired mutational, binding, and kinetic studies on complex formation between DPPIV (18) and ADA (31).

For wild type (WT) hADA nearly all residues on helix {alpha}2 were substituted, and the binding of the variants to WT rabbit DPPIV (rDPPIV) was studied in terms of dissociation constants (KD) and binding kinetics (kon, koff) (19). The affinity of the complex between WT hADA and rDPPIV (KD = 17 nM) is ~300x stronger than between WT murine ADA (mADA) and rDPPIV (KD = 5,400 nM). Compared with WT hADA, the most significant effects of single point mutations performed on helix {alpha}2 of hADA were found with Glu139 -> Ala, Arg142 -> Ala, and Asp143 -> Ala, which showed an ~10- to 8-fold decrease in binding affinity to rDPPIV, with KD between 160 and 112 nM (31). In all cases, the kinetic constants for complex formation (kon) were comparable (kon ~2 x 104 M-1 s-1) and similar to that for WT hADA and rDPPIV, whereas those for complex dissociation differed by one power (koff ~2-4 x 10-3 s-1) compared with WT hADA, (koff ~4 x 10-4 s-1. Such behavior (nearly constant kon but differing koff in point-mutated protein complexes) suggests important contributions by hydrophobic interactions (32), which, besides the salt bridges and hydrogen bonds detailed above, must add considerably to hDPPIV·bADA binding. This agrees with the finding that the (hDPPIV·bADA)2 complex dissociates at low ionic strength, suggesting that hydrophobic amino acids are essential for binding (33).

As interfacial residues of mADA and hADA differ solely in positions 142 and 143 on helix {alpha}2 (Fig. 3), a chimeric mADA construct containing the human {alpha}2 helix (residues 126-143) would be expected to bind to rDPPIV in a similar manner as WT hADA. The expected complex, however, showed a ~35-fold reduced KD of 591 nM compared with ~17 nM for rDPPIV·hADA. This is not due to conformational changes of {alpha}2, as superimposed backbone atoms of helices {alpha}1 and {alpha}2 of free mADA (PDB code 2ADA [PDB] ) with (hDPPIV·bADA)2 yielded a root mean square deviation of 0.53 Å. Although this is not significant at the 3.0-Å resolution level, the differences in dissociation constants may be due to other substitutions in mADA compared with hADA. The (hDPPIV:bADA)2 model shows that two nonconserved residues in the binding region of bADA, Gln175 and Ser131, are located at ~5 Å to Asn338 of hDPPIV and exchanged for charged Lys175 and Asp131 in mADA. This change could lead to far reaching electrostatic effects, thereby reducing the binding affinity in the complex formed by rDPPIV and chimeric mADA.

hDPPIV binds with a hydrophobic surface to ADA (Fig. 5). Compared with rodent DPPIV, which does not bind to endogenous ADA, four residues in the ADA binding site of hDPPIV are not conserved in any of the two rodent sequences, as depicted in Fig. 3. These are the bulky and hydrophobic residues Leu294, Ile295, and Val341 in hDPPIV that are all replaced by Thr in rat and by Ala294, Arg295, and Thr341 in mouse DPPIV, respectively. The positively charged Arg336 in hDPPIV is replaced by Val336 and Thr336 in rat and mouse, respectively. The significance of Leu294 or Val341 for ADA binding affinity was shown by mutational and subsequent binding studies, which indicated that mutations Leu294 -> Ala or Val341 -> Ala in hDPPIV reduced binding affinity to bADA, and drastic replacements Leu294 -> Arg and Val341 -> Lys even abolished bADA binding. The mutation Ile295 -> Ser showed no detectable effect (18), probably because Ile295 is located at the outer interface region (Figs. 4 and 5). The introduction of these three bulky hydrophobic residues in hDPPIV increases the hydrophobicity of the interface (Fig. 5). This may indicate a directional shift to the evolution of strong ADA binding affinity to DPPIV in higher mammals contrasted to the loss of binding in rodents as proposed by Abbott et al. (18).

The fourth nonconserved residue in the ADA binding site of hDPPIV is Arg336, which is replaced for Val and Thr in rat and mouse DPPIV, respectively (Fig. 3). Arg336, the only charged residue in the hDPPIV interface, forms a salt bridge to bADAAsp127 (see Figs. 4 and 5) and might therefore improve binding affinity.

ADA Binding Would Interfere with Tetramer Formation of DPPIV—Because the (hDPPIV·bADA)2 complex does not show significant structural changes compared with the isolated components hDPPIV and bADA, and their enzymatic activities are not affected (28), any modulation of a DPPIV-mediated signal transduction across the membrane that is induced by ADA binding may consequently be associated with yet unidentified sterical interference.

In this context, porcine DPPIV is of interest because it shares 88% amino acid sequence identity with hDPPIV and crystallized as a tetramer forming a " " -shaped complex (9). This is associated with the four {beta}-strands Asn279-Gln286 that form two antiparallel {beta}-sheets, thus extending the propeller blades IV to two eight-stranded {beta}-sheets, and the dimer-dimer interactions are augmented by the outer {beta}-strands of blades V (9). These segments (indicated by violet arrows in Fig. 1) share a homologous sequence in hDPPIV (Fig. 3), and although Gln286 is engaged in bADA binding (Fig. 4B), tetramer formation of DPPIV and binding of ADA are mutually exclusive. With this view, a recent study is explained that shows that hDPPIV promotes adhesion of lymphocytes to human epithelial cells, and that adhesion is significantly reduced by incubation with exogenous ADA (11). This suggests that ADA could regulate cell-cell adhesion mediated by DPPIV, although no evidence has yet been provided that DPPIV directly participates in cell-cell adhesion.

The proposed key role of the glycosylation state of Asn279 in preventing or enabling ADA binding (9), however, can be ruled out. This is because (hDPPIV·bADA)2 shows electron density for two N-acetylglucosamine residues attached to each Asn281 (corresponding to Asn279 in the porcine sequence) at an ~15 Å distance to the bADA surface (indicated by an asterisk in Fig. 1B), which is too far to directly interfere with ADA binding.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1W1I [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* These studies have been supported by DFG-Sonderforschungsbereich 449 and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table. Back

To whom correspondence may be addressed. Tel.: 49-30-838-53412; Fax: 49-30-838-56702; E-mail: saenger{at}chemie.fu-berlin.de.

|| To whom correspondence may be addressed. Tel.: 49-30-8445-1544; Fax: 49-30-8445-1541; E-mail: hua.fan{at}charite.de.

1 The abbreviations used are: DPPIV, dipeptidyl-peptidase IV; ADA, adenosine deaminase; hADA, human ADA; bADA, bovine ADA; mADA, murine ADA; hDPPIV, human DPPIV; pDPPIV, porcine DPPIV; rDPPIV, rabbit DPPIV; PDB, Protein Data Bank; r.m.s., root mean square; WT, wild type; TIM, triose-phosphate isomerase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Jörg Dobers for initial work, to Melanie Leddermann and Sabine Stehling for excellent technical assistance, to the staff at BESSY II (Berlin, Germany) for help with x-ray data collection, and to Dr. Timm Maier for help with structure determination. We are also grateful to Dr. Christoph Weise for N-terminal sequencing of DPPIV.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Mentlein, R. (1999) Regul. Pept. 85, 9-24[CrossRef][Medline] [Order article via Infotrieve]
  2. Hanski, C., Huhle, T., Gossrau, R., and Reutter, W. (1988) Exp. Cell Res. 178, 64-72[CrossRef][Medline] [Order article via Infotrieve]
  3. Gorrell, M. D., Gysbers, V., and McCaughan, G. W. (2001) Scand. J. Immunol. 54, 249-264[CrossRef][Medline] [Order article via Infotrieve]
  4. von Bonin, A., Huhn, J., and Fleischer, B. (1998) Immunol. Rev. 161, 43-53[CrossRef][Medline] [Order article via Infotrieve]
  5. Gutheil, W. G., Subramanyam, M., Flentke, G. R., Sanford, D. G., Munoz, E., Huber, B. T., and Bachovchin, W. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6594-6598[Abstract/Free Full Text]
  6. Valenzuela, A., Blanco, J., Callebaut, C., Jacotot, E., Lluis, C., Hovanessian, A. G., and Franco, R. (1997) J. Immunol. 158, 3721-3729[Abstract]
  7. Rasmussen, H. B., Branner, S., Wiberg, F. C., and Wagtmann, N. (2003) Nat. Struct. Biol. 10, 19-25[CrossRef][Medline] [Order article via Infotrieve]
  8. Thoma, R., Loffler, B., Stihle, M., Huber, W., Ruf, A., and Hennig, M. (2003) Structure (Camb) 11, 947-959
  9. Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H. U., and Brandstetter, H. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5063-5068[Abstract/Free Full Text]
  10. Lambeir, A. M., Diaz Pereira, J. F., Chacon, P., Vermeulen, G., Heremans, K., Devreese, B., Van Beeumen, J., De Meester, I., and Scharpe, S. (1997) Biochim. Biophys. Acta 1340, 215-226[CrossRef][Medline] [Order article via Infotrieve]
  11. Gines, S., Marino, M., Mallol, J., Canela, E. I., Morimoto, C., Callebaut, C., Hovanessian, A., Casado, V., Lluis, C., and Franco, R. (2002) Biochem. J. 361, 203-209[CrossRef][Medline] [Order article via Infotrieve]
  12. Wilson, D. K., Rudolph, F. B., and Quiocho, F. A. (1991) Science 252, 1278-1284[Abstract/Free Full Text]
  13. Kameoka, J., Tanaka, T., Nojima, Y., Schlossman, S. F., and Morimoto, C. (1993) Science 261, 466-469[Abstract/Free Full Text]
  14. Schrader, W. P., West, C. A., Miczek, A. D., and Norton, E. K. (1990) J. Biol. Chem. 265, 19312-19318[Abstract/Free Full Text]
  15. Martin, M., Huguet, J., Centelles, J. J., and Franco, R. (1995) J. Immunol. 155, 4630-4643[Abstract]
  16. Ludwig, K., Fan, H., Dobers, J., Berger, M., Reutter, W., and Bottcher, C. (2004) Biochem. Biophys. Res. Commun. 313, 223-229[CrossRef][Medline] [Order article via Infotrieve]
  17. Dong, R. P., Tachibana, K., Hegen, M., Munakata, Y., Cho, D., Schlossman, S. F., and Morimoto, C. (1997) J. Immunol. 159, 6070-6076[Abstract]
  18. Abbott, C. A., McCaughan, G. W., Levy, M. T., Church, W. B., and Gorrell, M. D. (1999) Eur. J. Biochem. 266, 798-810[Medline] [Order article via Infotrieve]
  19. Richard, E., Arredondo-Vega, F. X., Santisteban, I., Kelly, S. J., Patel, D. D., and Hershfield, M. S. (2000) J. Exp. Med. 192, 1223-1236[Abstract/Free Full Text]
  20. Dobers, J., Zimmermann-Kordmann, M., Leddermann, M., Schewe, T., Reutter, W., and Fan, H. (2002) Protein Expression Purif. 25, 527-532[CrossRef][Medline] [Order article via Infotrieve]
  21. Otwinowski, Z., and Minor, W. (1997) in Macromolecular Crystallography Part A (Carter, C. W., Jr., Sweet, R. M., eds) Vol. 276, pp. 307-326, Academic Press, New York[CrossRef]
  22. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022-1025[CrossRef]
  23. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  24. CCP4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  25. Jones, T. A. (1978) J. Appl. Crystallogr. 11, 268-272[CrossRef]
  26. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
  27. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524[Medline] [Order article via Infotrieve]
  28. De Meester, I., Vanham, G., Kestens, L., Vanhoof, G., Bosmans, E., Gigase, P., and Scharpe, S. (1994) Eur. J. Immunol. 24, 566-570[Medline] [Order article via Infotrieve]
  29. Aertgeerts, K., Ye, S., Shi, L., Prasad, S. G., Witmer, D., Chi, E., Sang, B. C., Wijnands, R. A., Webb, D. R., and Swanson, R. V. (2004) Protein Sci. 13, 145-154[CrossRef][Medline] [Order article via Infotrieve]
  30. Lo Conte, L., Chothia, C., and Janin, J. (1999) J. Mol. Biol. 285, 2177-2198[CrossRef][Medline] [Order article via Infotrieve]
  31. Richard, E., Alam, S. M., Arredondo-Vega, F. X., Patel, D. D., and Hershfield, M. S. (2002) J. Biol. Chem. 277, 19720-19726[Abstract/Free Full Text]
  32. Murrell-Lagnado, R. D., and Aldrich, R. W. (1993) J. Gen. Physiol. 102, 977-1003[Abstract/Free Full Text]
  33. Tsai, C. J., Lin, S. L., Wolfson, H. J., and Nussinov, R. (1997) Protein Sci. 6, 53-64[Medline] [Order article via Infotrieve]
  34. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Protein Eng. 8, 127-134[Abstract/Free Full Text]
  35. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. D. Desrosiers, K. M. Cembrola, M. J. Fakir, L. A. Stephens, F. M. Jama, A. Shameli, W. Z. Mehal, P. Santamaria, and Y. Shi
Adenosine Deamination Sustains Dendritic Cell Activation in Inflammation
J. Immunol., August 1, 2007; 179(3): 1884 - 1892.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Escusa, D. Laporte, A. Massoni, H. Boucherie, A. Dautant, and B. Daignan-Fornier
Skp1-Cullin-F-box-dependent Degradation of Aah1p Requires Its Interaction with the F-box Protein Saf1p
J. Biol. Chem., July 13, 2007; 282(28): 20097 - 20103.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H.-J. Lee, Y.-S. Chen, C.-Y. Chou, C.-H. Chien, C.-H. Lin, G.-G. Chang, and X. Chen
Investigation of the Dimer Interface and Substrate Specificity of Prolyl Dipeptidase DPP8
J. Biol. Chem., December 15, 2006; 281(50): 38653 - 38662.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. Pacheco, J. M. Martinez-Navio, M. Lejeune, N. Climent, H. Oliva, J. M. Gatell, T. Gallart, J. Mallol, C. Lluis, and R. Franco
CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse
PNAS, July 5, 2005; 102(27): 9583 - 9588.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. A. Weihofen, J. Liu, W. Reutter, W. Saenger, and H. Fan
Crystal Structures of HIV-1 Tat-derived Nonapeptides Tat-(1-9) and Trp2-Tat-(1-9) Bound to the Active Site of Dipeptidyl-peptidase IV (CD26)
J. Biol. Chem., April 15, 2005; 280(15): 14911 - 14917.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/41/43330    most recent
M405001200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weihofen, W. A.
Right arrow Articles by Fan, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weihofen, W. A.
Right arrow Articles by Fan, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement