Porphobilinogen deaminase and its structural similarity to the bidomain binding proteins

Porphobilinogen

deaminase the bidomain

and its structural binding proteins

Gordon

similarity

to

V. Louie

University of London, London, UK The tetrapyrrole-biosynthesis enzyme porphobilinogen deaminase has in two of its domains the same a/j3 polypeptide-chain fold as the transferrins and the periplasmic binding proteins. These proteins have in common an active-site cleft located at the interface between two domains. The binding proteins and porphobilinogen deaminase are likely related by divergent evolution. The interdomain motions observed in the binding’ proteins are suggested also to have an important role in the mechanism of the reaction catalyzed by porphobilinogen deaminase. Current Opinion

in Structural

Introduction The enzyme porphobilinogen deaminase (PBGD) is the third enzyme in the biosynthetic pathway of tetrapyrroles, which include the hemes, chlorophylls and corrins - macrocyclesof vital biological importance (reviewed in [l]). PBGD catalyzes a unique reaction involving the stepwise, head-to-tail polymerization of four molecules of porphobilinogen to assemble the openchain tetrapyrrole, hydroxymethylbilane (Fig. 1). PBGD occurs ubiquitously; the proteins from various organisms are monomeric, have sizes in the range 3445kDa, and haverelatively high amino acid sequenceconservation (at least32%among proteins from bacteria, fungi, plants and mammals). PBGD contains a novel dipyrromethane cofactor, assembled by the apoenzyme from two molecules of the substrate porphobilinogen and covalently attached to a cysteine side chain of the protein. The cofactor serves as a primer to which the product polypyrrole is finked

Biology 1993, 3:401-408

during the tetrapolymerization reaction, but is not itself turned over. Stepwise elongation of the polypyrrole generates four covalent enzyme-intermediate complexes, ESl-ES4 The ES4complex contains a bulky and highly acidic hexapyrrole (each pyrrole ring bears two carboxylate side-substituents), from which the product hydroxymethylbilane is hydrolyzed. Each of the ring-coupling reactions involves two steps: deamination of the incoming porphobilinogen substrate; and nucleophilic attack, by the terminal ring of the polypyrrole chain, on the carbon atom from which the ammonia was lost on the substrate molecule. Both of these processes are promoted by the capacity of the pyrrole nitrogen to feed electrons into the pyrrole ring [2]. The ability of PBGD to stabilize a partial positive charge at the pyrrole nitrogens of the two reacting pyrrole rings plays a key role in enzymatic catalysis. The recent crystal-structure determination of PBGD (from Escbendk coli) [3] has shown quite unexpect-

Potphobilinogen

corrins

Hydroxymethylbilane

Abbreviation PBGD-porphobilinogen

Fig. 1. The reaction catalyzed by PBCD. Four molecules of the substrate porphobilinogen are deaminated and linked head-to-tail. The product hydroxymethylbilane is the precursor to all tetrapyrrolic macrocycles, including the hemes, chlorophylls and corrins.

deaminase.

@ Current Biology Ltd ISSN 0959440X

401

402

Sequences and topology

edly that this enzyme has the same polypeptide-chain fold as ptqins~from two classes of binding proteins, the transferrins and the group-B periplasmic receptors (for recent reviews on these binding proteins, see 1451). This review&cusses the inferences that can be drawn from this structural similarity with a view to both the molecular evolution of these proteins and the mechanism by which PBGD catalyzes its intriguing reaction. The polypeptide-chain

fold of PBGD

’

The polypeptide chain of PBGD is folded into three a# domains (Figs 2 and 3). Domains 1 and 2 have a similar overall topology: a rearranged, doubly-wound, parallel P-sheet of five strands. Each sheet has four parallel and one antiparallel strands, with the initial wind containing two p-a units, and the second wind one p-a-p unit into which the antiparallel jW,rand is inserted. The a-helical segments flank each face of the sheet and are oriented essentially parallel to the ~-strands. The polypeptide-chain segment connecting strands g31 and p41 contains two conserved prolines and does not form the expected ahelix. The carboxy-terminal domain 3 is an open-faced, three-stranded parallel p-sheet with three a-hekes covering one face. Domains 1 and 2 can be considered to result from the dupiication’of a motif comprising a sheet of four parallel fi-strands with intervening a-helices, followed by an additional separate strand (also oriented parallel to the preceding four). Duplication of this motif about an approx-

Fig. 2. Stereo ribbon representation

imate twofold axis interdigitates the isolated strand into the dyad-related motif, thus yielding the mixed P-sheet in each domain. Note that the antiparaflel strand @51) in domain 1 is preceded by the entire domain 2. Thus, ’ the two domains are connected by two polypeptide-chain segments, and together these segments constitute an interdomain hinge. The active-site cleft is located at the interface between domains 1 and 2. The dipyrromethane cofactor is bound within this cleft; its side-substituent carboxylate groups form extensive salt-bridge and hydrogen-bond interactions with the polypeptide chain. The cysteine side chain to which the cofactor is covalently linked occurs on domain 3. The side-chain carboxylic acid of Asp84 interacts with the pyrrole nitrogens of both rings of the cofactor, and is identied as the key enzymatic group acting in the catalysis of the ring-coupling reactions. The polypeptidechain segment spanning residues 45-62 is poorly defined in the structure, and may constitute a mobile lid for the active-site cleft. Structural proteins

similarity

of PBCD to the binding

Domains 1 and 2 of PBGD resemble structurally a number of binding proteins, which also have a core of two topologically similar domains. These binding proteins include both of the duplicated lobes of the transfen-ins (lactoferrin [6,7] and serum transferrin [S] >,

of the polypeptide-chain backbone of PBCD. j3-Strands are drawn as broad arrows, a-helices as ribbons, and coil regions as thin rope. Domain 1 occurs at the lower left of the figure, domain 2 at the top, and domain 3 at the right. Also shown are the dipyrromethane cofactor and side chains that interact directly with the cofactor. C-ter, carboxyl terminus; N-ter, amino terminus.

Porphobilinogen

deaminase

and the bidomain

binding

proteins

Louie

Fig. 3. Schematic representation of topology and nomenclature of the secondary structural elements of PBCD. Helices (barrels) here are drawn as cylinders and are shaded if positioned behind the plane of the P-sheet (arrows).

and also the group II periplasmic receptors [9] (the sulfate[lo], phosphate- [ 111, maltodextrin- [9] and lysine/arginine/omithine-binding [ 121 proteins). All of these proteins share the same connectivity within the two rearranged parallel-a/P domains and have two interdomain hinge segments.Also similar are the lengths of corresponding secondaty structural elements, and the overall twists of the core P-sheets.In addition, a P-bulge occurs in strand /341in both PBGD and the transferrins. The results of the superposition of the C, backbones of PBGD and the binding proteins are shown in Fig. 4 and Table 1. The domains 1 of these proteins share the greatest structural similarity; there is no significant sequence identity ( < 12%) between PBGD and the other proteins. The five residues Glu-Asn-Arg-Ala-Aspfrom PBGD and the amino-terminal lobe (N-lobe) of lactoferrin, shown in Fig. 4(c), constitute the longest segment of identical sequence between PBGD and any of the binding proteins. There is signiiicant variability among these proteins in three regards: the lengths of loops connecting the alternating P-strandand a-helical segments;the precise lateral positioning of the a-helices with respect to the faces of the P-sheets;and the relative orientation of domains 1 and 2 within each protein molecule. In general, the loops are longer in the binding proteins than in PBGD, particularly in domain 2, with the notable exception of the mobile lid positioned in front of the active-site cleft in PBGD. The observed shifts in the positions of the a-helices serve to position optimally the ends of the helices for making ligand-binding interactions in the various proteins. As discussedfurther below, the third point (above) reflects the interdomain flexibility inherent in these proteins, which is likely to be of considerable mechanistic

importance in both the binding proteins and PBGD. An additional structural difference is that the extracellular transferrins contain several disulfide bonds, whereas the petiplasmic binding proteins contain few, and cytosolic PBGD contains none. A polypeptide-chain segment of variable conformation follows the core of domains 1 and 2 (i.e. after helix a41) in both PBGD and the binding proteins. In several of the binding proteins, this segment associates with the preceding two domains to form one or more additional interdomain connections; the lysine/arginine/omithine-binding protein lacks this segment entirely. In PBGD, domain 3 follows immediately after helix a41, joined through a single, short connecting segment.The polypeptide-chain fold of this domain has not been observed previously as an isolated unit, although a similar overall topology has been observed in a portion of E. coli Rl endonuclease deriving from two discontinuous segments [ 131. Functional homologies binding proteins

among

PBGD -and the

A common feature of PBGD and the binding proteins is a binding cleft formed at the interface between the two domains. The amino terminus of one (a12) or more a-helices is involved in forming the ligand-binding site. For PBGD and several of the other proteins, the ligand is oxyanionic. Sulfate- [lo] and phosphate-binding [ 111 proteins provide highly specific and tight binding sites for their respective, completely desolvated oxyanions, In the transferrins, the initial step in iron ligation is the binding of an essential carbonate anion, which

403

404

Sequences and topology

(b)

Fig. 4. Superposition of the C, backbones of domains 1 and 2 of PBGD with (a) the amino-terminal lobe of lactoferrin, and (b) maltodextrinbinding protein. PBCD is shown in thicker lines, with every tenth C, labelled. The locations in the interdomain cleft of the dipyrromethane cofactor (DPM) in PBCD, the iron (Fe) and carbonate (CO,) ligands in lactoferrin, and the maltose (Mal) ligand in maltodextrin-binding protein are also shown. Note that domains 1 and 2 of each of the binding proteins have been superposed independently onto those of PBGD; after the initial superposition of domain 1, the additional transformation required for superposition of domain 2 is a rotation by the indicated angle around an axis (dashed line) lying roughly parallel to the interdomain hinge strands. (c) Residues 69-77 in PBCD (thick lines and upper-case labels) and residues 48-56 in the amino-terminal lobe of lactoferrin (thin lines and lower-case labels). Hydrogen bonds’formed by side chains of these polypeptide-chain segments are shown as dashed lines (P, PBCD; L, lactoferrin). The superposition is based on the entire domain 1 of the two molecules, and not solely on the residues shown.

provides two coordinate bonds for the ferric ion that is subsequently bound [14]. The transferrins can also bind, in place of carbonate, other anions that possess a carboxylate group, such as oxalate [15]. In addition, in iron-free apotransferrin, the binding site is occupied by a chloride anion. In PBGD, the active-sitecleft must provide binding sitesfor up to 12 carboxylate groups carried on the side substituents of the dipyrromethane and the poiypyrrole product.

Despite the lack of sequence homology among PBGD and the binding proteins, several functionally important residues do occur at roughly equivalent positions. These residues include: a serine that both caps the al2 helix and hydrogen bonds to an acid oxygen on the ligand; an arginine near the amino terminus of this helix which interacts ionically with the oxyanion; and an aspartateon the loop joining j331and a31 which hasdiverse roles (see Table 1). Notably, the replacement of all three of these

Porphobilinogen

Table

1. Structural

comparison

of PBCD and the bidomain

binding

deaminase

and the bidomain

binding

Louie 405

proteins

proteins.

Proteina

PEG0

Structural

Lfn N-lobe

Lfn C-lobe

Tfn N-lobe

MBP

SBP

PBP

Carbonate,

Carbonate,

Carbonate,

Maltodextrin,

Sulfate

Phosphite

ferric

ferric

characteristics Carboxylate

L&and

groups

side on

ferric

ion

ion

ion

maltose

porphobilinogen Binding

al,. all. Ser129

helices

al2

serine

al,

arginine

~3,/a3,

Arg131

aspartate

Role of aspartate Slrwhwal with

(14

dl.

a12, a22

a&. al2 G

(1’2

(1%

01%

Thrl17

Thr461

Thrl20

Serl30

Ser139

Arg121

Arg465

Arg124

Arg134

Arg135 Asp56

Asp84

Asp60

Asp395

Asp63

ASP65

Asp66

Catalysis

Coordinates

Coordinates

Coordinates

Hydrogen-bonds

Salt-bridges

Hydrogen-bonds

Arg134

phosphate

iron

iron

iron

sugar

210

113

111

113

103

0.0

1.8

1.9

1.9

2.0

hydroxyl

superposition

PBCD

Number

of equivalent

Root mean in C, position

‘Coordinate

square

&s deviation

-

(A)

entries

in Brookhaven

1MBP. Lf”, human lactoferrin; rabbit serum transferrin.

Protein

Data

Bank 1301: human

MBP, maltodextrin-binding

protein;

lactoferrin,

1LFC; rabbit

PBCD, porphobilinogen

residues is thought to cause the loss of iron-binding activity in the carboxy-terminal lobe (C-lobe) of human melanotransferrin [16]. In addition, replacement of the serine (SerI30) in sulfate-binding protein causes a large increase in the dissociation constant of the sulfate ligand [171. In agreement with the much larger size of the dipyrromethanecofactor and the tetrapyrrolic product, the binding cleft in PBGD has a considerably larger volume than those in the binding proteins. (This comparison applies to all proteins presumably in their closed conformation, see below.) The one exception is maltodextrin-binding protein, which can bind a polysaccharide spanning seven glucosyl units [9]. Implications of the structural similarities with the binding proteins for the mechanism of PBGD

The four ring-coupling reactions catalyzed by PBGD in forming the hydroxymethylbilane product occur within an. active-site cleft which is partially occluded from the external medium, and involve the repeated use of a single catalyticsite (Asp84). Therefore, considerable flexibility in the vicinity of the active-site cleft is likely required to permit entry of the porphobilinogen substrate, release of the product, and repositioning of the growing polypyr role chain after each ring-coupling step, such that the terminal ring is disposed for reaction with the next molecule of substrate.The location of the active site at the interface between two domains clearly provides a major source of this flexibility [ 181. The binding proteins demonstrate two modes of such interdomain flexibility. First, in a number of these proteins, both open and closed conformations have been observed crystallographically, the two conformations differing in the width of the interdomaln cleft. Cleft widen-

serum

transferrin,

deaminase;

amino-terminal

PBP, phosphate-binding

lobe,

1TFD; maltodextrin-binding

protein;

SBP, sulfate-binding

protein; protein:

Tfn,

ing arises primarily from hinging about the two connecting interdomain strands, which results in rotation about an axis perpendicular to these strands (Fig. 5). In the amino-terminal lobe of apolactoferrin, exposed, basic side chains in the open cleft might serve to attract the carbonate ion; in lactoferrin, a 54” relative rotation of the two domains closes the cleft around the bound ligands and assembles the coordination sphere for the ferric ion [13]. In the group I periplasmic receptors, which are structurally related to the group II receptors (see below), open and closed conformations also differ by approximately 30” [ 191. The importance of interdomain motion in sulfate-binding protein has been confirmed by site-directed mutagenesis; an engineered disulfide bond cross-linking the two domains severely inhibits the releaseof bound sulfate ligand [20], whereas the abolition of interdomain salt bridges increases the rate of release

ml. A second form of interdomain flexibility is a twisting or swivelling about an axis parallel to the connecting strands. .Sharff et al - [22] have described the ligand-induced conformational change occurring in maltodextrin-binding protein as a 35” hinge bending (of the type described above) coupled with an 8’ interdomain twist. This twisting is also evident less directly in the differing relative orientations of domains 1 and 2 among PBGD, serum transferrin, lactoferrin, and maltodextrin-binding protein (see above and Fig. 4). In PBGD, residues ptimarily from domain 1 form the catalytic (Asp84) and substrate-binding sites,whereas residues primarily from domain 2 form both the binding site for the cofactor and an internal cavity that might serve to accommodate the growing polypyrrole chain. Therefore, a relative twisting of the two domains in PBGD provides an attractive model for the mechanism by which the terminal ring of the growing chain is carried into the appropriate position to react with the next molecule of porphobilinogen.

406

Sequences and topology

Fig. 5. Open (thick lines) and closed (thin) conformations of the binding proteins: (a) The amino-terminal lobe of lactoferrin, and (b) arabinose- and leucinelisoleucinelvaline-binding proteins. In each case, domain 1 has been superposed. Then, a rotation of domain 2, by the indicated angle around an axis roughly perpendicular to the plane of the figure, closes the interdomain cleft to sequester the ligand. A ribbon representation of the open form and the rotation axis are shown on the right-hand side of the figure.

Quiocho and colleagues [5,21] have emphasized the role of the biding proteins in excluding solvent from the vicinity of the bound &and in the closed binding cleft. An analogous function in PBGD can also be expected to be important, both for protecting reactive intermediates from exposure to solvent and for modulating the nature of the active-site environment. In PBGD, this function involves not only cleft closure, but also movement of the mobile lid. This lid, which is absent in all of the biding proteins, is proposed to close over the active-site cleft upon substrate binding [ 31. Unlike the binding proteins, PBGD lacks an a-helix in the segment of poiypeptide chain corresponding to a31. The amino end of an a-helix here would be pointed directly at the ring-coupling site, and could disfavour the positive charges that develop on the pyrrole nitrogens of the two reacting rings.

Evolutionary

origin

of PBCD

The close structural resemblance among PBGD and the binding proteins suggests that these proteins are related by divergent evolution. It is notable that site-directed mutants of Asps4 retain a small amount of activity (PM Jordan, personal communication), presumably because two porphobilinogen moieties that are held in the appropriate relative positioning can undergo spontaneously the rather facile ring-coupling chemistry. Thus, it can be speculated that the PBGD enzyme originated from a two-domain binding protein specific for an anionic, porphobilinogen-like or perhaps dipyrrolic &and, and then recruited catalytic groups and possibly the mobile lid. This ancestral PBGD might’have r&mentally catalyzed the coupling of porphobilinogen molecules within its binding cleft without covalently attaching the first porphobifinogen to the polypeptide chain. Covalent

Porphobilinogen

deaminase

&main

A-mminc

3

proteins

Louie

II

Monomeric I/ binding motif

I

Group II bidomain binding proteins

Fig. 6. Proposed evolutionary

relationship

among the group I and group II periplasmic

attachment could have arisen later when the carboxy-terminal domain was acquired. In addition, the polypyrrole product might initially have been attached directly but reversibly to the cysteinyl sull’hydryl group, and subsequently the first two pyrrole rings became permanently incorporated into the protein as the dipyrromethane cofactor. Evolutionary proteins

binding

Addition of

Duplication nf

and the bidomain

relationships

among

the binding

Additional evolutionary relationships become apparent if the internal duplication of domains 1 and 2 in PBGD and the binding proteins is considered. Quiocho and colleagues [9] have previously compared the group I and group II periplasmic binding proteins, which are structurally quite similar. These two groups are distinct in that the former construct the cores of each of their two domains from a pure doubly wound parallel P-sheet. In these group I proteins, the duplication of domains is clearly head-to-tall, and functionally analogous proteins (flavodoxin [23] and CheY [24] > representing the putative monomer are known. In PBGD and the group II binding proteins, the nature of the duplication is less obvious, because the duplicated motif comprises not an intact domain, but a four-stranded g-sheet together with an additional isolated strand, and domain 2 thus apparently occurs within rather than after domain 1 (as discussed above and in [lo] >. One model that accounts for the co~ectivily occurring in PBGD involves a rearranged, doubly wound, g-sheet motif, in which the helix normally following strand 4 is replaced by a reverse hairpin, causing strand 5 to be inserted antiparallel between strands 3 and 4. In a head-to-tail duplication of this motif, the first strand 5 might, rather than inserting into the preceding four-stranded sheet, become the nucleating, antiparallel strand in a second sheet; the second strand 5 would then become the linal antiparallel strand in domain 1. Alternatively, these strand rearrangements may have occurred directly within a binding protein of the group I type. A protein having the topology of a putative, monomeric precursor to the group II proteins has yet to be observed, although domain 3 of pyruvate kinase [25] and a portion of the eight-stranded g-sheet of dihydrofolate reductase [261 are close matches.It is however interesting that proteolytically generated, quarter-

1

binding proteins,

of lobes

PBGD and the transferrins.

sized fragments of transferrin (i.e. representing a single domain) have native-like folds and are capable of binding iron [27]. A common feature in all of the proteins discussed here (and typical of most parallel a,@ proteins [28]) is that in each individual domain, the carboxyl termini of the pstrands and the amino termini of the antiparallel a-helices are involved in forming a binding site. In the group I proteins, in which all of the strands have the same orientation, the two duplicated domains have a relative orientation appropriate for cooperatively forming a binding cleft only if their connecting segment traversesthe entire height of the second domain. The first P-strand in the second domain can then be directed toward the central cleft. In contrast, in PBGD and the group II binding proteins, the appropriate orientation of domain 2 is attained more efficiently, as each interdomain connecting segment is followed directly by a strand that runs antiparallel to the others in its domain. Therefore, the differing topologies of the group I and group II binding proteins represent alternative means of forming a binding cleft from duplication of a single binding motif. The proposed evolutionary relationships are summarized in Fig. 6. Acknowledgements I thank ail members of T Blundell’s laboratory for invaluable assistance; and aLso P Lindky, EN Baker, R Sarra and P Kuser for informatIve discussions on the transferfins. The figures in this paper were generated with the program SETOR [29], written by S Evans. The author is supported by a postdoctoral fellowship from the Medical Research Council of Canada,

References

and recommended

reading

Papers of particular Interest, published WithIn the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.

WARREN MJ, SCOTTAI: Tetrapyrrole Assembly and Moditicati?n into the Ligands of Biologically Functional Cofactors. Trends Bio&m Sci 1990, 15:4B6A91.

2.

PICHONC, CLEMENS KR,JACOBSADN AR, Sco-rr Al: On the Mechanism of Porphobiiogen Deaminase: Design, Synthesis, and Enzymatic Reactions of Novel Porphobilinogen Analogs. Teh-abedmn 1992, 254687-4712.

407

408

Sequences and topology 3.

4.

LCIUIEGV, BROWNUEPD, IAMBERTR, COOPERJB, WOOD SP, BLUNDEIL Tl, WARRENMJ, WOODCOCKSC, JORDANPM: Structiue of PorphobIIogen Deaminase Reveals a Flexible Multidomain Polymerase with a Single CataIytic Site. Nufuute 1992, 3593339. BAKEREN, IJNDIEY PF: New Perspectives on the Structure and Function of Transferrins. J Inorg Biocbem 1992, 47:147-160.

18.

SCHULZ GE: Domain Motions Biol 1991, 1:883-B%.

19.

SACK JS, SAPER MA, QUKXHO FA: PerIplasmic Binding Protein Structure and Function: ReEned X-Rav Structures of the LeucinellsoleucIneAine-Binding Pro&n and Its Complex with Leucine. J MO/ Biol 1989, 206:171-191. JACOBSON BI HE JJ, VERMERSCH PS, LEMONDD, QUIOCHOFA: Eneineered InterdomaIn Disuffide in the Periplasmic Receptoifor Sulfate Transport Reduces Flexibility. J Biol &em 1991, 266:5220-522s. JACOBSON BL, HE JJ, LEMONDD, QUIOCHOFA: Interdomain Salt Bridges Modulate Ligand-Induced Domain Motion of the Sulfate Receptor Protein for Active Transport. J MO/

20.

5.

QUIOCHOFA:Atomic Structures and Function of Periplasmic Receptors for Active Transport and ChemotaxIs. Cups Opin Struct Biol 1991, 1:922-933.

21.

6.

ANDEW~NBF, BAKERHM, NORRISGE, RICEDW, BAKEREN: Structure of Human L!+ctofenin: Crystallographic Structure Analysis and Refinement at 2.8A Resolution. J Mot Biol1989. 209:71l-734.

22.

BAKIZR EN, RuhmMLSV, ANDERSON BF: Transferrins: Insights into Structure and Function from Studies on Lactoferrin. Trends BiocJxm Sci 1987. 12:3SO-353.

8.

SARRA R, Garr R, @XINSKYB, JHOTl H. LINDI!ZYP: HighResolution X-Ray StudIeSon Rabbit Serum Transferrin: Preliminary Structure Analysis of the N-Terminal Hti-Molecule at 2.3k Resolution. AC& Crystallogr [B] 1991, 46763-771.

23.

9.

SPURUNO J,

Lu G.Y, QUKXHO FA: The 2.3-A Resolution Structure of the Maltose- or Maltodextrin-Binding Protein, a Primary Receptor of Bacterial Active Transport and Chemotaxis. J Eiol Cbem 1991, 266:5202-5219.

24.

10.

PFLUGRA’IH JW, QUIOCHOFA The 2A Resolution Structure of the Sulfate-BIIdIng Protein Involved in Active Transport in Salmonella typhimurium JMol Bioll988, 200:163-180.

11.

LUECKE H, QUKYZHO FA:

12.

KANGC-H, SHINW-C, YAMAGATA Y, GOKCENS, AMESGF-L,KIWI S-H: Crystal Structure of the Lysine-. ArginIne-, Or&hIneBinding Protein LAO from Salmonella typhimurium at 2.7-A Resolution. J Biol Cbem 1991, 266:23893-23899.

13.

ROSENBERG JM: Structure and Function of Restriction Endonucleases. Cur-r Opin Slrucf Biol 1991, 1:104-113.

14.

ANDESONBF, BAKERHM, NORRISGE, RUMBAUSV, BAKER EN: ApolactoferrIn Structure Demonstrates LiEand-Induced Conformational Change in Transferrins. Nature 1990, 344~784-787.

15.

16.

BAKER EN,

BAKER HM,

SMITH CA,

STEBBINS MR,

KAHN M,

HELLSTROM KE, HEUST~OM I: Human Melanotransferrin (p97) Has Only One Functional Iron-Binding Site. FEB.5Leti 1992, 298:215218. 17.

2s.

26.

27.

28.

29.

SHONGWE MS, SMITH CA, AINSCOUGH EW, BAKER HM, BRODIE

AM, BAKER EN: Anion BImdIIg by Human Lactofenin: Results from Crystallographic and Physicochemical Studies. Biochemistry 1992, 31:44514458.

HE JJ, QUIO~HOFA: A Nonconservative Serine to Cysteine Mutation In the Sulfate-Binding Protein, a Transport Recep tor. Science 1991, 251:147%1481.

Curr

Opin

Struct

Biol 1992, 223~27-30.

7.

High Specificity of Phosphate Transport Protein Determined by Hydrogen Bonds. Nature 1990, 347~402-406.

in Proteins.

30.

SW AJ, RODSFIHLE, SPUWNO JC, QUKXHO FA: Crystallographic Evidence of a Large Ligand-Induced Hinge-Twist Motion between the Two Domains of the MaItodexuin Binding Protein Involved in Active Transport and Chemotaxis. BiocbemWy 1992, 31:10657-10663. SUIIH WW, BURNETTRM, DAIUINGGD, LUDWIGML: Structure of the Semiquinone Form of FIavodoxIn from Cfostridium mp.:’ Extension of 1.8A Resolution and Some Comparisons with the Oxidized State. J lMo/ Biol 1977, 117:195-225. STOCKAM, MOITON~NJM, STOCK JB, SCHWT CE: ThreeDimensional Structure of CheY, the Response Regulator of Bacterial Chemotaxis. Nature 1989, 337745-749. I.!%NEM, MUIRHEADH, STAM~IER~ DK. STUARTDI: Structure of Pyruvate Kinase and Similarities with Other Enzymes: Possible Implications for Protein Taxonomy and Evolution. Nalwe 1978, 271:626-630. BOUNJT, FILMANDJ, MAITHEWS DA, HAMUNRC, KRALIT J: Caystal Structures of &chericbia cob and Lactobacillus casei Diivdrofolate Reductase ReEned at 1.7A Resolution. J Blol Cbefn 1982, 257:1365@13650. LINDLIZY PF, BAJAJM, EVANSRW,GARRA~T RC, HASNAIN SS,JHOTI H, KUS~RP, NEUM, PATELK, Sa R ET..IL:The Mechanism of Iron Uptake by Transfenins: the Structure of an 18 kd NII-Domain Fragment at 2.3A Resolution. Acla Cgstallogr [D] 1993, 49:292-304. BRANDEN C-l: The TIM Barrel-the Most Frequently Occurring Folding Motif in Proteins. Curr Opin SIrucl Biol 1991, 1:978-983. EVANS SV: SETOR: Hardware Lighted Three-Dimensional Solid Model Representations of Macromolecules. J MO/ Grapbiu 1993, in press. BERNSTEIN FC. KOEIZLETF. W~~lwhisGJB, MEYEREF JR, BIUCE MD. Roffi~rs JR, KENNA~UI 0, SHI~.~OUCHIT, TASUMIM: The Protein Data Bank: a Computer-Based Archival File for Macromolecular Structures. J MO/ Biol 1977, 112:53+S42.

GV Louie, Iaboratov of Molecular Biology, Department of Clystallography, Birkbeck College, University of London, MaIet Street, London WClE 7HX, UK.