The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation

Research Article

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The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Matthew J Pugmire and Steven E Ealick* Background: Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. In lower organisms (e.g. Bacillus stearothermophilus) PYNP accepts both thymidine and uridine, whereas in mammalian and other higher organisms it is specific for thymidine (designated thymidine phosphorylase, TP). PYNP shares 40% sequence similarity (and presumably significant structural similarity) with human TP, which has been implicated as a growth factor in tumor angiogenesis. It is thought that TP undergoes a major conformational change upon substrate binding that consequently produces an active conformation. Results: The crystal structure of PYNP from B. stearothermophilus with the substrate analog pseudouridine in its active site has been solved to 2.1 Å resolution. This structure confirms the similarity of PYNP to TP and supports the idea of a closed active conformation, which is the result of rigid body movement of the α and α/β domains. The active-site cleft, where the pyrimidine and phosphate substrates bind, is between the two domains. The structure reveals an asymmetric dimer in which one subunit is fully closed and the other is only partially closed.

Address: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. *Corresponding author. E-mail: [email protected] Key words: domain movement, gliostatin, plateletderived endothelial cell growth factor, thymidine phosphorylase Received: 3 July 1998 Revisions requested: 12 August 1998 Revisions received: 10 September 1998 Accepted: 25 September 1998 Structure 15 November 1998, 6:1467–1479 http://biomednet.com/elecref/0969212600601467 © Current Biology Ltd ISSN 0969-2126

Conclusions: The closed conformation of PYNP serves as a good model to better understand the domain movement and overall function of TP. Active-site residues are confirmed and a possible mechanism for substrate binding and subsequent domain movement is suggested. Potent inhibitors of TP might have significant therapeutic value in various chemotherapeutic strategies, and the structure of PYNP should provide valuable insight into the rational design of such inhibitors.

Introduction Pyrimidine nucleoside phosphorylase (PYNP) from the thermophilic bacteria Bacillus stearothermophilus is a dimer in solution with a subunit molecular weight of 46 kDa, and is reported to catalyze the phosphorolysis of both uridine and thymidine [1]. PYNP shares 40% sequence identity with human thymidine phosphorylase (TP) and thus serves as a good model to better understand the structure–function relationship of this intriguing enzyme/growth factor. Kinetic studies report that TP follows a sequential bi-bi mechanism where the order of substrate binding and product disassociation varies among species [2–5]. Detailed structural information of TP has so far come from the crystal structure of the Escherichia coli TP [6,7]. Each subunit of the TP dimer consists of a large mixed α-helical and β-sheet domain (α/β domain) separated from a smaller α-helical domain (α domain) by a large cleft. The active site of each subunit consists of a pyrimidinebinding site in the α domain and a phosphate-binding site across the cleft in the α/β domain. The distance between the phosphate- and pyrimidine-binding sites previously reported [6] is too large (e.g. 8–9 Å) for catalysis to occur

unless the α and α/β domains move together. Similar domain movements have been reported in other enzymes [8,9]. The domain movement proposed for TP would close the active-site cleft and bring the substrates into mutual proximity. Evidence of this proposed rigid body movement of the two domains has been reported for the E. coli TP where the two domains have moved under packing constraints of different space groups [7]. Modeling studies have suggested a hypothetical closed-form TP structure, similar to the structure of PYNP reported here. In order to confirm the hypothesis of a closed-cleft active conformation, a noncleaveable substrate analog, pseudouridine, was co-crystallized with PYNP. The 2.1 Å resolution crystal structure reveals a fully closed enzyme conformation with pseudouridine and phosphate bound in the active site. As previously hypothesized [7], the two domains have come together via rigid body movement to close the active-site cleft. Details of the conformational change should facilitate a better understanding of the mechanism of domain closure, and of the subsequent catalysis. Such information is vital for developing effective inhibitors that could have therapeutic value.

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Results and discussion Structure determination

The crystal structure of PYNP was determined with molecular replacement techniques using E. coli TP as a search model. PYNP has the same fold as that reported for E. coli TP [6], containing identical secondary structural features, with the exception of a three-stranded antiparallel β sheet in the α/β domain, which has not been reported previously. The strands of this new β sheet, labeled β1C–β3C, along with the previously reported secondary structural features (seventeen α helices, labeled H1–H17; a sixstranded mixed β sheet, labeled β1A–β6A; and a fourstranded antiparallel β sheet, labeled β1B–β4B) are shown in Figure 1a. A topology diagram is shown in Figure 2, which also shows the arrangement and labeling of the secondary structures; the residues involved in these secondary structures are listed in Table 1. The residues that make up the α and α/β domains, as well as three interdomain connecting loops are also similar to those reported for E. coli TP. It has been proposed that the three loops connecting the α and α/β domains (labeled L1–L3) act as hinges that allow the domains to move as rigid bodies. The only major structural difference between PYNP and the previously reported E. coli TP structure is in the relative positions of the α and α/β domains with respect to each other.

The asymmetric unit of PYNP is composed of a dimer, but the two subunits (referred to as A and B) are unrelated by a noncrystallographic two-fold axis. Instead, subunit B is in a fully closed conformation with phosphate and pseudouridine bound in the active site and subunit A, in contrast, is in a more open conformation (only phosphate was found in the active site; see Figure 1b). The asymmetric dimer structure is probably a result of the packing of the dimer in the unit cell; the symmetry-related portions of subunit A would prevent the α/β domain of subunit A from rotating into the closed conformation, whereas subunit B has no such restrictions. Alternatively, communication between subunits might result in an asymmetric structure where one subunit has high substrate affinity and the other has low affinity. As a consequence, this unique conformation provides structural evidence of two distinct states along the pathway of cleft closure. Comparing these two PYNP states with the structures of E. coli TP [7] provides a more complete view of the progression of domain movement, which is depicted in Figure 3. Several of the 433 amino-acid residues in each subunit showed poor density throughout the building and refining process. Thr154 and Gly155 of subunit A, which are located in hinge L2, were not visible in the 2Fo–Fc or Fo–Fc maps.

Figure 1 (a)

(b) H1

N

100 280 140 80

H3 H2

H9

H10

6A 3C H6

1C 2C3B 1B 2B 4B

380400 340 420 240 360

C5A 4A 1A 2A H7 H12 3A H5

H11

20

N′ 40 40′

N

20′

160′ 360′ 180′ 60′ 400′ 220′ 240′120’ 340′ 200′ 380′ 420’ C′ 80’140′

H14 H13 H15

320′ 280′ 260′ 100′ 300′

100 280 140 80

300 320 260

120 200

380 400

340 420 240 360

220 60 160 180

220 60 160180

H8

H17

120 200

H4

H16

300 320 260

20

N′ 4040′

N

20′

160′ 360′ 180′ 60′ 400′ 220′ 240′ 340′ 120’200′ 38′′ 420’ C′ 80′ 140′ 320′ 280′ 260′ 100′ 300′ Structure

(a) Ribbon drawing of a single subunit of PYNP from B. stearothermophilus. The α/β domain is red, the α domain is blue, and the three hinge regions (L1–L3) are green. The secondary structural elements are labeled. The crystallographically observed phosphate and pseudouridine with the modeled ribosyl group are shown as ball-andstick models to indicate the position of the active site. (b) A stereoview

of the Cα trace of the functional dimeric PYNP using the same coloring scheme as in (a). Subunit A has every 20th residue labeled 20–420 and subunit B has every 20th residue labeled 20′–420′. The crystallographically observed substrates (two phosphate ions and the pyrimidine ring of pseudouridine, as well as the modeled ribose moiety) are shown. This figure was produced using MOLSCRIPT [39].

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

Figure 2

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Table 1 Residues involved in the secondary structrual elements of B. stearothermophilus PYNP.

N

H2

H3

H1

H4

α Helices

L1

H8

H9

L2

H14

L3

H13

H12 β6A β2A

β3A

β1A

H5

H6

H7

β4A

β5A

C

H10 H11

H15 β4B

β-Sheet C

1A: 78–83 2A: 105–111 3A: 147–151 4A: 195–200 5A: 213–237 6A: 429–432

1B: 245–248 2B: 334–338 3B: 376–379 4B: 395–398

1C: 344–348 2C: 385–388 3C: 416–418

β3B

H16

β1C β3C

β-Sheet B

β1B

β2B

H17

H1: 3–12 H2: 18–30 H3: 35–47 H4: 52–65 H5: 91–101 H6: 119–126 H7: 135–145 H8: 159–169 H9: 176–191 H10: 210–227 H11: 249–260 H12: 265–283 H13: 288–302 H14: 303–315 H15: 321–325 H16: 351–361 H17: 404–414

β-Sheet A

β2C Structure

Topology diagram showing the arrangement of the secondary structural elements in PYNP. Helices are represented as circles and β strands are represented as triangles. Secondary structural features in the α domain are blue whereas those in the α/β domain are red. The interdomain loops are green. This figure was produced using TOPS (http://www3.ebi.ac.uk/tops/).

The flexibility in this region of the protein (particularly Gly152 and Gly155) is probably an important factor in domain movement. The corresponding loop of subunit B is slightly better defined, although Thr154 was again not visible. Asp324 is located in a loop on the surface of the protein, and is not visible in the map of subunit B. In addition, there are 12 sidechains in subunit A and 26 sidechains in subunit B that are either partially visible or not visible at all in the electron-density maps. In all cases these residues are on the surface of the protein and apparently lack a single stable conformation. Both subunits A and B showed density that suggests a metal ion is adjacent to the phosphate-binding site. In addition, two MES molecules (MES was the buffer used during crystallization [10]) are bound to the surface of the dimer. Domain movement

Using HINGEFIND [11] and VMD [12] to analyze the differences between subunits A and B resulted in the

partitioning of the protein into two rigid-body domains as shown in Figure 4. These two domains correspond to the α and α/β domains, with the exception of parts of H10, that have been partitioned as part of the α domain by HINGEFIND. These results are supported by similar findings in the analysis of domain movement in E. coli TP that suggests the α and α/β domains move rigidly with the exception of H10, which showed some independent movement from the rest of the α/β domain [7]. When comparing subunit A to subunit B of PYNP, HINGEFIND determined that the major portion of the α/β domain rotates 21° around an axis that is oriented as shown in Figure 4. HINGEFIND was also used to analyze the degree of PYNP domain movement when compared with the three models of E. coli TP. It appears that subunit A of PYNP has undergone partial domain closure when compared to the three models of E. coli TP. In particular, subunit A has an additional 15° rotation beyond the tetragonal crystal form, which is the most closed of the E. coli TP structures. This partial closure in subunit A could result from partial equilibrium with pseudouridine or could result entirely from crystal-packing contacts that lock the subunit in the open state. This unique structure, when compared with the three different structures of E. coli TP, provides a view of the enzyme in several intermediate states along the trajectory of cleft closure (Figure 3). Important interdomain contacts between the α and α/β domains are shown in Table 2. Comparing the contacts present in subunits A and B identifies residues that are potentially important in stabilizing the closed conformation. Most of these contacts involve residues in the loop

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Figure 3 Stereo diagram showing Cα traces of the PYNP and E. coli TP models [7] compared with the most open E. coli TP model (the monoclinic spacegroup) to show the progression of domain closure: (a) orthorhombic E. coli TP (green) compared to monoclinic E. coli TP (red), (b) tetragonal E. coli TP (blue) compared to monoclinic E. coli TP (red), (c) PYNP subunit A (cyan) compared to monoclinic E. coli TP (red) and (d) PYNP subunit B (gold) compared to monoclinic E. coli TP (red). This figure was produced using MOLSCRIPT [39].

(a)

(b)

(c)

(d)

Structure

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

1471

Figure 4 Stereoview of the orientation of the rotation axis determined by HINGEFIND [11] when comparing subunits A and B of PYNP. The model has been partitioned into rigid domains that are shown in blue (corresponding to the α domain) and in red (corresponding to the α/β domain). The three interdomain loops are green, and the orientation of the domain rotation axis is shown in black. This figure was produced using MOLSCRIPT [39].

Structure

region from residues 363–374. This surface loop extends from the α/β domain across the cleft to the α domain where it acts as a latch to hold the two domains together. Ala366, Lys367 and Lys368, which are responsible for four interdomain contacts, are at the tip of this loop. The positive charge (here provided by lysine residues) is highly conserved among the known TP sequences in this loop region [10]. Additionally, there are two highly conserved glycine residues at the start of this loop region

that might provide necessary flexibility during domain movement. Four of the contacts involving residues from the surface loop in subunit B are formed with residues from the α domain from subunit A. This strongly suggests a functional role for the dimer structure in closing the active-site cleft. In the open-cleft form (subunit A), there is a hydrogen bond between the sidechain of Asn175 and the backbone of

Table 2 Comparison of interactions across the active-site cleft between the a and a/b domains in each of the subunits (A and B) of B. stearothermophilus PYNP. Subunit A

Subunit B

α Side

α/β Side

Type of interaction

α Side

α/β Side

Type of interaction

N175 K188

F207 D80

A56 T59† M60 V174 I177 I180 A181 I184 M185 K188† I189

T84† I196 L198 V200 F207 M208 L217 V220 M221 I224 V228 T232†

Hydrogen bond Salt bridge, hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket

D169 Y36 (of A*) Y165 Q37 (of A*) D5 (of A*) D35 D32 (of A*) K188

G115 K368 R112 A366 W360 K368 K367 D80

M60 V174 I177 I180 A181 I184 M185 K188† I189

T84† L114 I196 L198 V200 F207 M208 L217 V220 M221 I224 V228 T232*

Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Hydrogen bond Salt bridge Salt bridge Salt bridge, hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket Hydrophobic pocket

*The dimer interface is such that interactions between the other subunit occur across the cleft. †Indicates that the aliphatic portion of this polar sidechain is involved in the hydrophobic pocket.

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Figure 5

Tyr165 (Herringbone stacking)

Arg168

Schematic drawing of the active-site contacts seen in subunit B of PYNP. Hydrogen bonds are shown as dashed lines and distances are indicated in Å. The position of the ribose moiety, not observed experimentally, has been modeled. The drawing was produced using CHEMDRAW (CambridgeSoft, Cambridge, MA).

NH

NH2

+

NH2

2.8

O NH

OH

3.2 NH

3.3

OH

HO

Ser183

O O

2.9 NH3+

Lys187

OH N HO

2.5

Ser83

3.1 N H

NH O

2.8

His82 NH3+

P O

2.9

O

3.1

O

Lys81

HO

2.9 2.8

2.6

H2O

Ser110

NH3+

HO

Lys108 Thr120 Structure

Phe207 that is not present after the cleft has closed. In subunit B, the sidechain of Asn175 has flipped to a different rotamer position after the cleft has closed and does not appear to be involved in any hydrogen bonds. The reformation of this bond in subunit B could be important for cleft re-opening when catalysis is complete. The large hydrophobic region between the two domains is formed largely by the interface between H4 and H9 from the α domain and H10 from the α/β domain. This hydrophobic pocket is at the opposite side of the cleft from where the substrates are likely to enter and exit, and does not appear to undergo significant change upon domain movement.

The active site

Important interactions of the substrates with residues in the active site are shown in Figure 5. The phosphatebinding site is located in the α/β domain at the carboxyterminal end of β-sheet A between strands β1A and β2A. Residues from β1A, β2A and H6 that are important in phosphate binding include Lys81, Ser83, Lys108, Ser110 and Thr120. The hydrogen-bonding pattern that occurs in the phosphate-binding site of subunit A is identical to that of subunit B, with the exception of two water molecules that form hydrogen bonds with O4 of the phosphate ion in subunit A but are absent in subunit B.

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

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Figure 6 Stereo diagram of the metal-binding site showing the contacts made with surrounding residues. This figure was produced using MOLSCRIPT [39].

L243

L243 G88

G88 2.3

2.3

2.5

3.0 2.7

2.5

3.0 M 2.7

M 2.6

T90

2.6

T90 A246

E255

A246

E255 Structure

In both subunits A and B there is also an apparent metalion-binding site, adjacent to the phosphate-binding site, that is located in a loop between β5A and β1B. This unidentified metal ion has a coordination number of five, forming interactions with the backbone carbonyl oxygen atoms of Leu243, Ala246 and Gly88, and the sidechains of Glu255 and Thr90 (see Figure 6). The metal peak height in the Fo–Fc density map is consistent with a Ca2+ or K+ ion. The most common coordination number for Ca2+ is seven, although a Ca2+ coordination number of five has been observed previously [13]. In addition, the range of bond lengths are not consistent with standard Ca2+binding modes. It is more likely that the metal is K+ because the protein was stored in potassium phosphate buffer. No other metals were explicitly present during the purification and crystallization procedures. If the metal is something other than K+, it likely originated in the E. coli cells during protein expression and would require high binding affinity to remain bound to PYNP throughout the purification and crystallization process. What role this metal ion plays in the function of PYNP, if any, has yet to be determined. The pyrimidine-binding site is on the α-domain side of the cleft and is located between helices H8 and H9. Key contacts with the pyrimidine ring are formed with residues from these two helices, in particular Arg168, Ser183, Tyr165 and Lys187. In subunit A, where the active site is partially exposed to solvent, the electron-density maps show weak density in the pyrimidine-binding site that could represent a water molecule, or possibly pseudouridine with low occupancy. Because the density did not definitively identify a pseudouridine or a water molecule, nothing has been modeled in this site. Subunit B however, which is fully closed, shows good electron density for the

uracil ring of pseudouridine, which forms hydrogen bonds as indicated in Figure 5. The ribose portion of pseudouridine, however, was not visible in subunit B in the density maps. The variability of the sugar pucker and rotational freedom around the glycosidic bond in the absence of stabilizing hydrogen bonds of the ribose moiety are likely the causes of this weak electron density. The absence of water molecules in the ribosebinding region of subunit B (particularly the two water molecules that hydrogen bond to O4 of the phosphate ion in subunit A) supports the argument that the ribose is present, but is not rigid enough to appear in the density maps. It is unlikely that the highly stable C–C glycosidic bond of pseudouridine is cleaved during crystallization. The conformation of the ribose moiety, predicted using molecular modeling techniques, is shown in Figure 5 and reveals several possible hydrogen-bonding contacts. Assuming the modeled position of the ribose moiety is accurate, the nucleoside binds in the β-conformation with a dihedral angle of the glycosidic bond (defined by O4′–C1′–N1–C2) of 165°, which is classified as +antiperiplanar according to the Klyne and Prelog convention [14]. The ribose moiety shows a C4′-endo sugar pucker that is similar to several purine nucleosides bound to purine nucleoside phosphorylase (PNP) [15,16]. The pyrimidine nucleoside is in an unusual, high-energy conformation [14] that would likely put strain on the glycosidic bond. Glycosidic bond strain due to a high-energy nucleoside conformation is consistent with the catalytic mechanism proposed for the phosphorolysis of purine nucleosides [16]. Of the known TP sequences, E. coli and human are reported to be specific for 2′-deoxyribosides, whereas PYNP from B. stearothermophilus and B. subtilis show no such

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specificity at the 2′ position of the ribose moiety. The specificity of the remaining sequences has not been reported. The only difference when comparing the positions of the active-site residues in the crystal structures of E. coli TP and B. stearothermophilus PYNP is Lys108 in B. stearothermophilus. The sidechain of Lys108 forms a hydrogen bond with O1 of the phosphate, which in turn forms a hydrogen bond with the 2′OH of the ribose moiety (see Figure 5). This lysine residue is highly conserved [10], except in the human and E. coli species where it is a methionine. The different chemical nature of the sidechains at this position among species could contribute to a different binding mode of the phosphate ion and subsequently affect the specificity at the 2′OH position. Another residue in the active site that might be important, but whose function is yet unknown, is His82. This histidine residue is positioned on the α side of the ribose ring near the 2′OH and C1′ positions and is completely conserved among the known sequences of TP and PYNP. The Nδ1 of His82 appears to be protonated and is in a good position to donate a hydrogen bond to the backbone carbonyl oxygen of Ser83. It is not clear from this structure what role His82 plays in the active site. If the imidazole ring were rotated 180°, the Nδ1 would be 2.8 Å from C1′ of the ribose moiety. Assuming Nδ1 was not protonated, this hypothetical position could help stabilize the positive charge that builds up at the C1′ position in the proposed transition state (see below). A histidine residue involved in a catalytic triad in the PNP active site has been implicated in the catalytic mechanism; it serves to deprotonate the substrate phosphate ion, making it a stronger nucleophile for purine nucleosides [16]. Although such a catalytic triad is not present in TP, another possible role for His82 could be to deprotonate phosphate after it binds (via Nδ1 of the imidazole ring), before the pyrimidine nucleoside binds. This would require significant rotation of the imidazole ring, possible because of the absence of any restraining van der Waals surface nearby. PYNP also shows no specificity for the 5-position of the pyrimidine ring. The environment near this position consists of a hydrophobic pocket formed by the sidechains of residues Phe207, Val174, Ile180 and Leu114. Due to the position of the phenyl ring of Phe207 near the 5-position of the uracil ring, it is unlikely that large substituents could be accommodated at this position. There appears to be enough room for a methyl group (as in the case of thymidine) but the sidechain of Phe207 might have to move slightly. In the case of the E. coli TP, the corresponding phenylalanine residue is flipped into a different sidechain conformation, which leaves room for a methyl group at the 5-position. This difference in sidechain conformation could be the result of domain movement however, and does not necessarily suggest specificity differences between E. coli TP and PYNP at the 5-position. The observation that Phe207 has

changed sidechain conformations could indicate conformational flexibility that might be useful in adapting to different substituents at the 5-position of the pyrimidine ring . Implications for the catalytic mechanism

Based on the structural information from this work, a probable mechanism of pyrimidine phosphorolysis is similar to the SN1-type reaction proposed for PNP [16] — the polarization of the N1–C1′ glycosidic bond of the nucleoside. This polarization is encouraged by three major factors: the nucleoside could bind in a high energy conformation (+antiperiplanar glycosidic dihedral angle and C4′-endo ribose pucker), which would strain the glycosidic bond; the flow of electrons from the glycosidic bond to the pyrimidine ring (presumably to O2 and O4) could be readily stabilized by the positive charges of Arg168 and Lys187; and the resulting partial positive charge at C1′ could be stabilized by the formation of an oxocarbenium ion at O4′, which in turn would be stabilized by the negative charge on O4 of the phosphate ion. Because O4 of the phosphate ion only forms one hydrogen bond (with the 3′OH of the ribose, based on modeling studies) and because it is the closest oxygen to the C1′ position of the ribose, it appears to be in a position to attack at the C1′ position of the pyrimidine nucleoside. Assuming the ribose moiety to be in the modeled position as shown in Figure 5, the distance between O4 and C1′ is 3.6 Å. This distance is consistent with the corresponding distances seen in several complexes of PNP [15]. Once the partial positive charge at the C1′ position has built up, the negatively charged O4 of the phosphate is then poised to attack, resulting in a pyrimidine base and α-ribose 1-phosphate as products. The cleaved pyrimidine base can then be protonated at N1 by water, or, alternatively, by His82. Figure 7 shows the positions of the active-site residues around the substrates, as well as a schematic diagram of a proposed catalytic mechanism. Although several possible active-site residues have been identified from this structure, as well as the structures of E. coli TP [6,7], it should be emphasized that further studies, including kinetic analyses and site-directed mutagenesis, are necessary in order to positively identify and clarify the specific functions of residues involved in the catalytic mechanism. Possible mechanism of domain movement

The studies of E. coli TP have suggested [7] that phosphate binding triggers partial domain closure through the formation of a hydrogen bond between the sidechain of His119 and the backbone carbonyl oxygen of Gly208. This hydrogen bond results in the rotation of the α/β domain by approximately 8°. An analogous hydrogen bond is apparent in both subunits A and B of PYNP between His116 and Gly205. The PYNP hydrogen bond and subsequent domain movement coincides with the binding of the phosphate ion and serves to order the loop region 112–117. A comparison of subunits A and B of PYNP suggest a possible mechanism of

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

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Figure 7 Proposed catalytic mechanism of PYNP. (a) Stereoview of the active-site residues that are observed crystallographically with the exception of the ribose moiety of the pyrimidine nucleoside, which has been modeled. (b) Schematic diagram of a possible catalytic mechanism for cleaving the glycosidic bond in a pyrimidine nucleoside. The figure was produced using MOLSCRIPT [39] and CHEMDRAW (CambridgeSoft, Cambridge, MA).

(a) R168

R168

S183

S183

Y165

Y165

K187 S83

K187 S83

H82 S110

W T120

H82 S110

W T120

K81

K81

K108

K108

(b) Arg168

H2N

+

Arg168

H2N

NH2

+

δ–

NH2

O

O H O

NH OH

OH

Ser183 N

O

Ser183

δ+

O

N

O +H N 3

HO –

OH

Lys187 –O

O–

O

Oδ

–

+H N 3

HO

OH

H O

NH

P

Lys187

O– P

O

O

OH

OH

[E•S]‡

[E•S]

Arg168

H2N

+

NH2 O H O

NH

Ser183 N H

OH

+H N 3

O O–

O

Lys187

P HO

O

OH

O

OH

[E•P]

Structure

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Structure 1998, Vol 6 No 11

Figure 8

α/β

T154 NH

Schematic drawing of a possible domain closure mechanism. The pyrimidine substrate is represented as a gray circle. The three hinge regions are green and are labeled L1–L3. The proposed hydrogen bond formation that forms a β turn is indicated by the dashed line in the L2 region.

α/β

L2 Q153 7.4 O

T154 NH

L1 O 3.2 O

NH3+

L157

Thd

L2

3.0

3.2 O

O

NH3+

L157 O

O D161

L1

3.2

L3

L3

Thd

NH 2

α

K187

D161

α

K187

Structure

further domain movement, triggered by the binding of substrate in the pyrimidine-binding site. In subunit A, Gln153 does not appear in the density maps and is presumably disordered due to flexibility. In subunit B however, when substrate is bound, Gln153 is visible in the density and forms a hydrogen bond with Asp161. The formation of this hydrogen bond apparently coincides with the formation of a β turn in residues 154–157 of the L2 hinge, where a hydrogen bond forms between the carbonyl oxygen of Leu157 and the backbone nitrogen of Thr154. Two glycine residues in L2, Gly152 and Gly155, likely play an important role in allowing torsional flexibility in this region. Several residues in this interdomain loop (Gly152, Gln153 and Asp161) are highly conserved among the known TP sequences [10].

Another observation from the comparison of subunits A and B is the herringbone stacking interaction of Tyr165 with the pyrimidine ring. When substrate binds, Tyr165 apparently forms a hydrogen bond with the carbonyl oxygen atom of Arg112 on the other side of the cleft, and possibly with the 5′-OH of the ribose. The sidechain of Tyr165 must change conformation slightly to make this interaction possible, which suggests it might also play an active role in facilitating and/or stabilizing the domain movement once the pyrimidine ring is introduced into the active site. In conjunction with the movement of Tyr165, Leu114 also changes conformation slightly as compared with subunit A where the hydrophobic sidechain now sits above the face of the aromatic ring of Tyr165.

The formation of a β turn in the L2 hinge region decreases the length of this interdomain loop (residues 152–158) and effectively serves to pull the α and α/β domains together as depicted in Figure 8. The other two interdomain loops (L1 and L3) are located near the opposite side of the active-site cleft and likely serve as passive hinges between the two domains as they move. When substrate binds in the pyrimidine-binding site, the sidechain of Lys187 apparently changes conformation so that the hydrogen bond with O2 of the pyrimidine ring (shown in Figure 5) can form. Prior to substrate binding (as in subunit A), Lys187 is in position to form a salt bridge with Asp161. Once this salt bridge is disrupted by substrate binding, Asp161 is then able to form a hydrogen bond with Gln153 (see above). The movement of Gln153 to form this hydrogen bond coincides with the movement of L2 necessary for the formation of the β turn from residues 154–157.

The mechanism involved in reopening the cleft likely begins with the weakening of the Lys187–O2 bond once the glycosidic bond is cleaved. When the pyrimidine ring is protonated at the 1-position following glycosidic cleavage, the charge that was built up on O2 and O4 now flows back to the 1-position. This reversal of charge flow weakens the two contacts involving Arg168, Lys187 and O2 and O4 of the pyrimidine ring. The sidechain of Lys187 can then flip back to reform the salt bridge with Asp161, which consequently will disrupt the Asp161– Gln153 hydrogen bond, which in turn weakens the 157– 154 reverse-turn hydrogen bond that helped pull the domains together. Breaking the Asp161–Gln153 hydrogen bond probably facilitates domain movement as the cleft reopens. Another possible contributing factor to the opening of the cleft is the steric hindrance between the product and His82. After phosphate attacks at the C1′ of

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

the ribose, the position of His82 probably inhibits the movement out of the active site of the phosphate–ribose product. This could force the sidechain of His82 to change conformation, which would in turn repel the sidechain of Ile184 (on the face of the α domain) and facilitate cleft opening.

Biological implications Mammalian species have two pyrimidine nucleoside phosphorylases (PYNPs) that are specific for catalyzing the reversible phosphorolysis of thymidine (thymidine phosphorylase, TP) and uridine (uridine phosphorylase). In contrast, other organisms (e.g. Bacillus stearothermophilus and B. subtilis) use a single PYNP that does not distinguish between uridine and thymidine and shares significant sequence identity with the TP of higher organisms. PYNP from B. stearothermophilus shares several significant features with human TP including high sequence identity (40%); the same quaternary structure (homodimer); and a similar molecular weight (92 kDa). These similarities suggest that structural studies of PYNP should provide a reasonable model for human TP. The substrates of TP (thymidine and phosphate ion) bind on either side of a large cleft between the smaller α-helical domain (α domain) and the larger mixed α-helical and β-sheet domain (α/β domain) and are separated by a distance of at least 8 Å [6]. Previous structural studies of E. coli TP hypothesized that the α and α/β domains undergo a large conformational change that closes the active-site cleft [6,7]. The data presented here support the hypothesis that TP undergoes a major conformational change upon substrate binding. This conformational change involves the rigid body movement of the α and α/β domains toward each other, closing the active-site cleft, and bringing the substrates into mutual proximity so that catalysis can proceed. The 2.1 Å resolution complex crystal structure of PYNP from B. stearothermophilus presented here, with the noncleavable substrate analog pseudouridine bound in the active site, shows the enzyme in the closed-cleft, active conformation. This model of PYNP gives detailed structural information of the active conformation that has not previously been observed. These structural details are critical to understanding the enzymatic action and overall function of PYNP. Because of the significant similarity between PYNP and human TP, these structural results provide valuable insight, by inference, into the structure–function relationship of an important enzyme/growth factor. The structure of PYNP also provides the first model of a phosphorylase that accepts both thymidine and uridine. Analyzing the differences between PYNP and TP should also help to elucidate the mechanisms of substrate specificity. The reversible phosphorolysis of pyrimidines is an important step in the salvage pathway where the free bases are

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used in nucleotide synthesis and the pentose 1-phosphates are further metabolized in the pentose phosphate pathway and subsequently used in glycolysis. Recent studies have shown that platelet-derived endothelial cell growth factor (PD-ECGF), which was initially characterized by its angiogenic and endothelial cell chemotactic activity, is identical to human TP [17]. Similarly, gliostatin, initially characterized by its ability to inhibit glial cell growth, which subsequently stimulates neuron growth, is also identical to human TP [18]. PD-ECGF shows angiogenic and endothelial cell chemotactic activity and gliostatin inhibits glial cell growth, which subsequently stimulates neuronal survival. TP expression is reported to be inducible by interferon and other cytokines [19,20], and high levels of TP are found in numerous types of tumor cells [21–24], probably a result of the TP growth factor activity. Whether or not the enzymatic activity of TP is directly related to these growth-factor activities has yet to be determined. Inhibition of the TP enzymatic activity, as well as modulation of thymidine levels in vivo have traditionally been important to numerous chemotherapeutic strategies [25–28]. The recent findings that link TP to growth-factor activities have increased the interest in its potential as a target for structure-based drug design. Detailed structural information of this enzyme should greatly aid the search for effective pharmaceutical agents that could be used in various chemotherapeutic strategies.

Materials and methods Purification, crystallization and data collection The cloning, purification and co-crystallization of B. stearothermophilus PYNP with the substrate analog pseudouridine has been reported previously [10]. After pseudouridine was added to the protein, which was stored in phosphate buffer, the crystallization conditions (0.1 M MES buffer at pH 6.0–6.2 and 25% PEG 6000) resulted in crystals suitable for X-ray diffraction. Pseudouridine is identical to the substrate uridine with the exception of the N1 position, which has been substituted with C and the C5 position which has been substituted with N. These substitutions result in a C–C glycosidic bond which is noncleavable, but leaves the key contacts of this substrate analog unchanged. A low temperature data set was collected at the Cornell High Energy Synchrotron Source (CHESS) at station A1 using a 1K ADSC CCD detector. Low temperature conditions (approximately –170°C) were provided by the use of a heat exchange cryostat from Molecular Structure Corporation, The Woodlands, TX [29]. The crystals were frozen for data collection by mounting directly from the hanging drop into a loop, then immediately placing the loop in the liquid nitrogen cold stream. No additional cryoprotectants were required to obtain good quality diffraction images. The crystals belong to the space group P21 with cell parameters of a = 53.6, b = 70.5, c = 122.8 and β = 98.0. Assuming two subunits in the asymmetric unit (a full dimer), the crystals have a Matthews number (VM) of 2.57 Å3/Da, and a solvent content of 52%. A total of 202° of data were measured. Each data frame consisted of a 1° oscillation measured for 20 s. Data were integrated and scaled using the programs DENZO and SCALEPACK [30]. The crystals diffracted to a maximum resolution of 2.0 Å, however the completeness beyond 2.1 Å was less than 50%. Data collection statistics are shown in Table 3.

Structure determination The structure was solved with molecular replacement techniques using E. coli TP in spacegroup P43212 [6] as the search model. Rotation

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Replacing all sidechains (other than glycine and alanine) with serine and performing positional refinement using data from 10 to 3 Å resulted in Rfree = 46.4% and R = 43.7%.

Table 3 Data collection and refinement statistics for B. stearothermophilus PYNP.

Model building and refinement Data collection Number of observations Number of unique reflections Resolution (Å) Completeness (%) Rsym (%) I/σ > 3 (%) Redundancy Refinement R (%) Rfree (%) RMS deviations from ideal geometry Bond (Å) Angle Dihedrals Impropers Ramachadran plot quality % In core allowed regions % In additional allowed regions % In generously allowed regions % In disallowed regions Total number of nonhydrogen atoms Protein Water Pseudouridine MES Metal ions (K+ or Ca2+ ) Average B factors (Å2) Mainchain Sidechain Water Ligands (substrates and metals)

396 276 55 716 30–2.0 (2.07–2.0)* 88.4 (41.7) 6.7 (23.2) 82.4 (58.5) 2.9 (1.6)

23.2 27.6 0.007 1.2 22.6 1.2 90.3 9.2 0.5 0 6287 110 8 24 2 25.5 28.4 25.4 35.3

*Numbers in parentheses refer to the highest resolution shell.

functions (consisting of a standard rotation function followed by Patterson correlation, PC, filtering [31]), and translation functions (consisting of rigid body refinement in a P1 unit cell, followed by the standard translation function), were carried out using the program X-PLOR [32]. Attempts to locate a noncrystallographic two-fold axis with self-rotation functions did not produce a convincing solution. Cross-rotation functions using data from 10 to 4 Å and a dimer as the search model were also unsuccessful. It was found, however, that if only a monomer was used as the search model, the rotation search was successful, in addition to the translation function, which resulted in the top peak being 10σ above the mean, 6.8σ above the second highest peak, a S/N [33] of 1.5 and R = 49.9. These results suggested that the two monomers of PYNP were somehow different in the unit cell, and were not related by an exact two-fold axis. The rotation and translation functions both improved (12.4σ above mean, 9.2σ above the second highest peak, S/N = 1.5, and R = 48.5%) upon using the monomer plus the α domain of the second subunit as the search model. Every attempt to place the final α/β domain of the search model into the unit cell, although giving an acceptable rotation function solution, made the translation function worse. Finally it was found that if the translation function was carried out using data from 12 to 5 Å, as opposed to 10 to 4 Å, that during the rigid body refinement the α/β domain of the second subunit rotated significantly after which the translation function improved (10.3σ above mean, 7.1σ above the second highest peak, S/N = 1.4, and R = 47.6%). Upon examining the model after this translation search, it was apparent that the α/β domain of the second subunit had rotated approximately 20° so as to produce a fully closed form, whereas the first subunit was still in a partially open conformation.

Model building was initially carried out by fitting the model to 2Fo–Fc and Fo–Fc electron density maps using data from 8.0 to 3.0 Å in the program O [34]. With the exception of several loop regions, the polyserine model fit the density maps quite well and a substantial number of the correct sidechains were built in. Following this initial manual rebuild of the model, the building process continued by carrying out cycles of positional refinement, simulated annealing and overall B-factor refinement using X-PLOR followed by manual rebuilding in the program O. Throughout the building and refinement process, 5% of the reflections were excluded in order to monitor the Rfree value. During the first five cycles of building, noncrystallographic symmetry (NCS) restraints were used (using a different symmetry operator for each of the α and α/β domains) starting with restraint values of 300 kcal/mol and gradually decreasing as judged appropriate by the Rfree value. During the sixth building cycle, releasing the NCS yielded a lower Rfree value that justified not using NCS restraints from then on. The resolution of the data was increased gradually during the building process, extending to 2.75 Å, then to 2.5 Å and finally to 2.3 Å. In each case, the resolution was not extended until further information could no longer be extracted from the current electron density maps. During several of the building cycles simulated annealing omit maps, as implemented in X-PLOR, were used to build poor regions of the model. After 11 cycles of building, refinement of the model was carried out in a similar manner using cycles of manual rebuilding in the program O followed by positional refinement, simulated annealing, and isotropic individual B-factor refinement with X-PLOR. During the refinement process, several ligands (phosphate, pseudouridine, and MES) were apparent from the density and were modeled near the end of the refinement process. Density indicative of a phosphate molecule was clearly visible in the phosphatebinding pocket of both subunits A and B. The uracil ring of pseudouridine was clearly seen in subunit B, but not in subunit A. After five refinement cycles at 2.3 Å, the resolution of the data was extended to a final value of 2.1 Å. Density representing MES molecules was noticeable in two locations near the surface of the protein. Molecules of MES were included in the model after the fifth cycle of refinement. In addition, a large peak in the density maps indicated the presence of an ion near the phosphate-binding site that was present in both subunits A and B. After modeling several metal ions in these positions, it was apparent that the peak size was consistent with a Ca2+ or K+ ion. Metal ions (either Ca2+ or K+) were included in the model after the fifth round of refinement. Protein sidechains that showed very weak or no electron density were not included in the model. Water molecules were added where peaks appeared in both the 2Fo–Fc and Fo–Fc maps and were in position to form good hydrogen bonds. After a total of ten refinement cycles, the model had the statistics shown in Table 3.

Modeling the ribose moiety In order to estimate the position of the ribose moiety of pseudouridine in the active site, QXP [35], which uses a Monte Carlo-based docking method, was employed. LAZYMOUSE (the graphical front end to QXP) was used to build the protein shell to which pseudouridine was docked. Starting with the refined PYNP coordinates of subunit B, all residues outside a 10 Å radius from the active site were cut away, followed by the addition of polar hydrogen atoms. This shell was then energy minimized using MACROMODEL v6.0 [36] where the nonhydrogen atoms were highly constrained using a constraint parameter of 500 kJ/mol. Two cycles (500 steps each) of steepest descent minimization using the Amber force field [37,38] resulted in a converged energy minimum. This shell was then used to dock the pseudouridine substrate using QXP. Several of the active-site residues (His82, Ser183, Lys187 and Arg168 ) were allowed to be constrained flexible during the energy minimization cycle of QXP docking. A total of 3000 steps of the full Monte Carlo docking method in QXP (named MCDOCK) resulted in the ribose position that is shown in Figure 5.

Research Article The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation Pugmire and Ealick

Structure analysis HINGEFIND [11] is a useful program for identifying effective rotation axes that describe domain movement when comparing protein structures. This method first identifies rigid domains between two structures by a least squares method, and then determines where the rotation axes are located, as well as the degree of rotation about these axes. This effective rotation can then be used to approximate the motion of a rigid body. The Tcl version of HINGEFIND was used in conjunction with VMD [12] to calculate the rotation parameters associated with the rigid body movement of the α and α/β domains of PYNP. When comparing subunits A and B of PYNP, a tolerance level of 1.5 Å was used. This resulted in the partitioning of the protein into two rigid domains (118 residues of the α domain and 293 residues of the α/β domain). A tolerance level of 1.8 Å was used in the comparison of PYNP to the three models of E. coli TP. This resulted in a similar partitioning of residues into the α and α/β domains as in the case of comparing subunits A and B of PYNP.

Accession numbers The coordinates for the PYNP model have been deposited with the Brookhaven Protein Data Bank with accession code 1brw.

Acknowledgements The authors would like to acknowledge the support of the W.M. Keck Laboratory for Molecular Structure, the Lucille P. Markey Charitable Trust and the National Institutes of Health training grant (# T2GM08500A). This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation under award DMR-9311772, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award RR-01646 from the National Institutes of Health.

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