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Journal ArticleDOI

The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica.

05 Apr 2006-Journal of Biological Inorganic Chemistry (Springer-Verlag)-Vol. 11, Iss: 4, pp 398-408

TL;DR: The crystal structures of AAP are reported at 0.95-Å resolution at neutral pH and at low pH to allow the precise modeling of atomic positions, the identification of theMetal bridging oxygen species, and insight into the physical properties of the metal ions.
Abstract: The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-A resolution at neutral pH and at 1.24-A resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.

Summary (3 min read)

Introduction

  • Bridged bimetallic enzymes contain two metal ions held in close proximity by a protein ligand and/or an oxygen atom, usually from bulk solvent, that spans two metal ions.
  • It has been proposed that both metal ions are necessary to recognize and bind substrate, to activate the attacking nucleophile, and to stabilize intermediates of the reaction.
  • A chemical reaction mechanism has been proposed for AAP [6] in which the substrate binds to AAP by first coordinating the carbonyl oxygen of the N-terminal amino acid to Zn1 followed by the coordination of the N-terminal amine to Zn2.
  • In the absence of electron density corresponding to hydrogen atoms, precise coordination distances can be used to establish the identity of the bridging water species by comparing its Zn-O bond distances.

Enzyme purification

  • All chemicals used in this study were purchased commercially and were of the highest quality available.
  • AAP was purified from a stock culture kindly provided by Céline Schalk.
  • Cultures were grown according to the previously reported protocol with minor modifications [6] to the growth media.
  • Purified enzyme was stored at −196 °C until needed.

Crystallization

  • The protein was crystallized using the conditions reported previously [11] with the exception that Tris was replaced with HEPES [12] .

Data collection and processing

  • An AAP crystal was removed from the hanging drop, soaked in mother liquor containing 10% glycerol for 1 min, coated with Paratone-N oil, mounted in a 0.5-mm Hampton Research CryoLoop, and flash-cooled to −173 °C in liquid nitrogen.
  • Second, the low-resolution data were collected by reducing the exposure time to 0.5 s, moving the 2θ angle to 0°, and increasing the sample-to-detector distance to 200 mm.
  • Refinement for both data sets was carried out using the software package CNS [14] followed by SHELX.
  • The structure was refined almost to convergence.
  • The blocks contained overlapping residues so that every ESD could be estimated with all contributing atoms being refined in at least one of the refinement cycles.

Results and discussion

  • The reaction mechanism step at which deprotonation of the bridging solvent molecule occurs, the most likely candidate to function as the nucleophile, is unknown.
  • Of the 184 peaks associated with carbon atoms, a modest increase in the number of potential C α hydrogen peaks was observed when increasing the resolution from 1.20 to 0.95 Å, while the number of peaks associated with side chain carbons and amide nitrogen atoms doubled (Table 2 ).
  • The normal motion of the heavier atoms that associate with hydrogen atoms can reduce the hydrogen contribution to background noise, making it impossible to observe them in a Fourier difference map and difficult to reproduce them from one data set to the next.
  • The calculated estimated standard deviation associated with each distance is shown in parentheses Electron density corresponding to potential hydrogen atoms in the active site was not observed in the Fourier difference map with coefficients F obs−F calc; however, the protonation state of the bridging oxygen species can be determined by comparing Zn-O distance standards found in the CSD [9] and those studied by computational methods [10] with the precise Zn-O distances obtained in this structure.
  • If the bridging oxygen species is OH − and Glu151 is the proton acceptor [17] , then the dangling oxygen of Glu152 may serve to stabilize the bridging hydroxide ion or the protonated carboxylate side chain by forming a hydrogen-bonding interaction with them.

Fig. 2. Schematic of the active site of native aminopeptidase from Aeromonas proteolytica (AAP)

  • For Asp179, there is a 0.04-Å difference in bond length between the two carbon-oxygen bonds of the side chain carboxylate, with the distance to the inner-sphere oxygen being slightly shorter than the dangling oxygen distance.
  • The carbon-oxygen distance to the inner-sphere oxygen of Asp179 is significantly shorter than the carbon-oxygen distances for the metal ligated oxygen atoms of Glu152 and Asp117 (Δd=0.05-0.07 Å) and is consistent with that for C=O distances measured in small molecules, implying that this bond has more double-bond character.
  • If the bridging oxygen in the native enzyme is an OH − , as identified earlier herein, then the level of precision obtained at high resolution should make it possible to observe the 0.1-0.2-Å change that should occur in Zn-O distances when the bridging oxygen changes its protonation state from OH − to OH2.
  • A coordination number change is the most likely cause since the Zn2-O distance remains consistent with that observed for Zn-OH, suggesting that the bridging solvent species has not taken on a second proton.
  • A result of the increase in the Zn1-OH bond distance is the strong Zn2-OH interaction (1.93 Å).

Movement of metal ions during catalysis

  • During the course of the enzymatic reaction, the distances of the metal ions to their ligands may change in accordance with the chemical state of the substrate/intermediate.
  • At 1.8-Å resolution, the resolution at which several crystal structures of AAP have been studied, it is impossible to assess the thermal motion of the atoms because of the isotropic treatment of the thermal parameters.
  • The anisotropic treatment of thermal motion has indicated that the active site is actually asymmetric with one metal binding site being more rigid than the other.
  • This relative increase in the thermal motion of the Zn1 nonbridging ligands can be attributed to the lack of possible protein hydrogenbonding partners for Glu152 and His256.
  • LPA coordinates to both zinc ions and pushes Zn1 0.60 Å away from its position in the native enzyme, while Zn2 remains close to its original position.

Insight into the chemical mechanism

  • The structures of AAP presented herein have provided additional evidence for the proposed reaction mechanism of AAP that was not obtainable from lower-resolution X-ray structures.
  • Glu151, the proton acceptor in this activation step [17] , donates a proton to the penultimate nitrogen of the product later in the reaction cycle.
  • On the basis of kinetics, spectroscopic and X-ray crystallographic data [6, 21] it has been proposed that substrate coordinates to the bimetallic center in a stepwise fashion with the carbonyl oxygen of the N-terminal amino acid first coordinating to Zn1 followed by the coordination of the free amine to Zn2 (Fig. 5 , species 2 and 3).
  • Spectroscopic evidence for Co(II)-substituted AAP indicates that the two metal ions in the AAP active site bind in a sequential fashion and that upon introduction of substrate the coordination geometry of the first metal binding site changes from four to five coordinate.
  • In addition, the bridging solvent molecule becomes terminal and is bound to the first metal binding site.

Fig. 5. Proposed chemical reaction mechanisms for AAP

  • On the basis of X-ray crystallographic and spectroscopic studies of AAP complexed with the substrate analog inhibitor 1-butaneboronic acid (BuBA) [21], the metal ion thought to play the catalytic role was identified as Zn1.
  • A molecule of water was also observed near the active site.
  • It may provide information about the differences in the behavior of the two metal ions in the nearly symmetrical AAP active site.
  • For intermediate 3a to form, the second substrate binding step would involve coordination of the N-terminal amine to Zn2 followed by breaking of the Zn2-OH bond, consistent with spectroscopic and X-ray crystallographic data.
  • Distinguishing the contributions of the two identical Zn(II) ions in AAP is an important question since AAP displays 80% of its total activity level with only a single metal ion bound.

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the Low pH Crystals of Aminopeptidase from
Aeromonas proteolytica
William Desmarais
Brandeis University
David L. Bienvenue
Utah State University
Krzysztof P. Bzymek
Utah State University
Gregory A. Petsko
Brandeis University
Dagmar Ringe
Brandeis University
See next page for additional authors
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Marquette University
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Chemistry Faculty Research and Publications/College of Arts and Sciences
This paper is NOT THE PUBLISHED VERSION; but the author’s final, peer-reviewed manuscript. The
published version may be accessed by following the link in the citation below.
JBIC Journal of Biological Inorganic Chemistry, Vol. 11, No. 4 (June 2006): 398-408. DOI. This article is ©
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Springer does not grant permission for this article to be further copied/distributed or hosted elsewhere
without the express permission from Springer.
The high-resolution structures of the neutral
and the low pH crystals of aminopeptidase
from Aeromonas proteolytica
William Desmarais
Program in Biophysics and Structural Biology, Brandeis University, Waltham
The Rosenstiel Basic Medical Sciences Research Center, MS029Brandeis University, Waltham
David L. Bienvenue
Department of Chemistry and Biochemistry, Utah State University, Logan
Krzysztof P. Bzymek
Department of Chemistry and Biochemistry, Utah State University, Logan
Gregory A. Petsko
The Rosenstiel Basic Medical Sciences Research Center, MS029Brandeis University, Waltham
Department of Chemistry, Brandeis University, Waltham
Department of Biochemistry, Brandeis University, Waltham
Dagmar Ringe
The Rosenstiel Basic Medical Sciences Research Center, MS029Brandeis University, Waltham
Department of Chemistry, Brandeis University, Waltham
Department of Biochemistry, Brandeis University, Waltham

Richard C. Holz
Department of Chemistry, Marquette University, Milwaukee, WI
Department of Chemistry and Biochemistry, Utah State University, Logan
Abstract
The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes
the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH
and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic
positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the
metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is
likely relevant to all binuclear metalloproteases.
Keywords
Crystallization, Electronic structure
Abbreviations
AAP Aminopeptidase from Aeromonas proteolytica
BuBA 1-Butaneboronic acid
CSD Cambridge Structural Database
ESD Estimated standard deviation
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
LPA l-Leucinephosphonic acid
rms Root mean square
Tris Tris(hydroxymethyl)aminomethane
The coordinates for the 0.95-Å resolution structure and the 1.24-Å structure at pH 4.7 were deposited in the RCSB Protein
Data Bank and have PDB ID numbers of 1RTQ and 2DEA, respectively.
Introduction
Bridged bimetallic (binuclear) enzymes contain two metal ions held in close proximity by a protein ligand and/or
an oxygen atom, usually from bulk solvent, that spans two metal ions. These enzymes catalyze diverse reactions
that include but are not limited to hydrolysis, isomerization, dehydration, and redox chemistry [1, 2, 3, 4
]. They
utilize most first-row transition metal ions and can be either homonuclear or heteronuclear. The physical
properties of the two metal ions determine their Lewis acidities and, in turn, regulate the activity of the
enzymes. Although the exact role of each metal ion during a given reaction cycle is not completely understood,
it has been proposed that both metal ions are necessary to recognize and bind substrate, to activate the
attacking nucleophile, and to stabilize intermediates of the reaction.
As a model system for bridged bimetallic enzymes, we have studied the extracellular, broad-specificity
aminopeptidase from Aeromonas proteolytica (AAP). AAP is a 30-kDa, monomeric enzyme that utilizes two
zinc(II) ions in its active site to remove N-terminal amino acids from peptides or proteins [5
]. A chemical reaction
mechanism has been proposed for AAP [6] in which the substrate binds to AAP by first coordinating the carbonyl
oxygen of the N-terminal amino acid to Zn
1
followed by the coordination of the N-terminal amine to Zn
2
. An
activated water molecule then attacks the scissile bond at the carbonyl carbon, resulting in the formation of a
gem diolate that is stabilized by interactions with both zinc ions. A conserved active-site residue, Glu
151
, accepts

a proton from the bridging water molecule and then transfers it to the penultimate amino nitrogen of the new N
terminus [7
]. Finally, the enzyme returns to its native state upon the release of products and the addition of a
new bridging water species. In this mechanism, the role of Zn
1
is to activate the nucleophilic water molecule
from H
2
O to OH
, to activate the carbonyl carbon of the substrate, and to position the nucleophile for attack on
the substrate. The role of Zn
2
is to assist in lowering the pK
a
of the bridging water molecule, to provide
enhanced specificity for and to orient N-terminal peptide substrates, and to stabilize intermediates in the
reaction pathway.
Although structural and spectroscopic studies have provided a great deal of evidence for the role of each metal
ion during the catalytic reaction cycle of AAP, many questions remain unanswered: Why are two metal ions
employed in this and most other aminopeptidases? What is the protonation state of the bridging solvent
molecule in the resting enzyme? When does the bridging solvent become activated to a nucleophile? What
changes in the enzyme are required to accommodate substrate binding and the various intermediate states? To
completely understand the role of each metal ion in AAP with respect to the catalytic reaction cycle, it is
essential to know the precise position of every atom in the active site, including those of the hydrogen atoms.
Two very important hydrogen atoms are those attached to the bridging oxygen. For AAP to perform the
hydrolysis step, the bridging water presumably must be activated from H
2
O to OH
. As a first step in the
determination of the protonation states of the metal ligands, the bridging oxygen, and Glu
151
in AAP, we have
determined the 1.20-Å resolution structure of native AAP at pH 8.0 in a tris(hydroxymethyl)aminomethane (Tris)
buffer [8
]. This structure showed a single molecule of Tris chelated to the two metal ions in the active site,
making it impossible to identify the protonation state of the bridging oxygen and the active-site amino acids.
Subsequently, we removed any interference caused by Tris by crystallizing the protein in 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer.
Here we present the 0.95-Å resolution structure of AAP in HEPES buffer, pH 7.5, and the 1.24-Å resolution
structure of AAP at pH 4.5 in acetate buffer. These high-resolution structures have led to a very precise analysis
of AAP’s active site, determination of the identity of the protonation state of the bridging oxygen, based on
bonding distances, assignment of the double-bond distribution for the active-site carboxylates, and observation
of a change in the coordination number of the proposed catalytic zinc ion at low pH, which may serve as a model
for the first step in the reaction pathway. In the absence of electron density corresponding to hydrogen atoms,
precise coordination distances can be used to establish the identity of the bridging water species by comparing
its ZnO bond distances. A survey of the Cambridge Structural Database (CSD) [9] and ab initio calculations [10
]
have provided ZnOH and ZnOH
2
bonding distances that should serve as standards for comparing the precise
bonding distances obtained in protein crystal structures determined at ultrahigh resolutions. The increased
quality of the electron density maps at 0.95-Å resolution has also allowed for a more detailed view of some
hydrogen positions, the solvent region, and electron distribution in the active-site than was possible at 1.20-Å
resolution.
Materials and methods
Enzyme purification
All chemicals used in this study were purchased commercially and were of the highest quality available. AAP was
purified from a stock culture kindly provided by Céline Schalk. Cultures were grown according to the previously
reported protocol with minor modifications [6
] to the growth media. Purified enzyme was stored at −196 °C until
needed.
Spectrophotometric assay
AAP activity was measured by monitoring the hydrolysis of 0.5 mM l-leucine p-nitroanilide [10 mM N-
tris(hydroxymethyl)methylglycine, pH 8.0] spectrophotometrically at 25 °C by monitoring the formation of p-
nitroaniline [6
]. The extent of hydrolysis was calculated by monitoring the increase in absorbance at 405 nm (Δε

Figures (8)
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  • ...A similar pattern of Glu-carbonyl interactions is observed in structures of the above-mentioned aminopeptidases (24,47-49) (Figure 6)....

    [...]

  • ...The authors carried out DFT calculations (using B3LYP functional) on the model of the active site of AAP (so-called cluster model), which was derived from an existing crystal structure of a substrate-free AAP (24) with a model dipeptide (Ala-Ala) docked as a substrate....

    [...]

  • ...Despite the above-mentioned efforts, the reaction mechanism of GCPII is not fully understood in detail, although some information can be derived from extrapolation of the known reaction mechanism of the related AAP, for which more experimental (X-ray crystallography, kinetics, spectroscopy, and site-directedmutagenesis) and computational data are available (24,25)....

    [...]

  • ...Our experimental and theoretical data on the GCPII reaction mechanism are consistent with findings of others on related aminopeptidases (2,24-26) and suggest a common mechanism of substrate hydrolysis by this class of enzymes....

    [...]

  • ...Similar distance variability of Znhas beennotedduring the reaction cycle ofAAP, and it has been suggested that positional flexibility of metal ions is required as a response to a constantly changing environment in the active site of binuclear enzymes (24)....

    [...]


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Book ChapterDOI
TL;DR: The program is designed to be easy to use and general for all space groups and uses a conventional structure-factor calculation rather than a fast Fourier transform (FFT) summation, which is much slower than programs written specifically for macromolecules.
Abstract: Publisher Summary SHELXL-93 was originally written as a replacement for the refinement part of the small-molecule program SHELX-76. The program is designed to be easy to use and general for all space groups and uses a conventional structure-factor calculation rather than a fast Fourier transform (FFT) summation. The latter would be faster but in practice involves some small approximations and is not suitable for the treatment of anomalous dispersion or anisotropic thermal motion. The price to pay for the extra precision and generality is that SHELXL is much slower than programs written specifically for macromolecules. This is compensated for, to some extent, by the better convergence properties, reducing the amount of manual intervention required. A new version, SHELXL-97, was released in May 1997; this is the version described in the chapter. The changes are primarily designed to make the program easier to use for macromolecules. Advances in cryogenic techniques, area detectors, and the use of synchrotron radiation enable macromolecular data to be collected to higher resolution than was previously possible. In practice, this tends to complicate the refinement because it is possible to resolve finer details of the structure. It is often necessary to model alternative conformations, and in a few cases, even anisotropic refinement is justified.

2,155 citations


Journal ArticleDOI
07 Nov 1996-Chemical Reviews
TL;DR: Zinc enzymology is, compared to some other current areas of metallobiochemistry, a maturing field, but in addition to further developments of structure-function relationships it has also provided a number of surprising new results and ideas in the last few years.
Abstract: Zinc enzymology is, compared to some other current areas of metallobiochemistry, a maturing field, but in addition to further developments of structure-function relationships it has also provided a number of surprising new results and ideas in the last few years. In fact, the number of studies makes it impossible to provide a comprehensive review of the recent literature on zinc enzymology here, and the authors therefore focus on those zinc enzymes for which structure-function relationships are possible on the basis of structural and biochemical data. This means that, with a few exceptions, only zinc enzymes for which NMR or crystal structures are available are included here. Another seemingly simple, yet experimentally sometimes complex issue concerns the choice of which metalloenzyme is a zinc enzyme. Since there is in principle no difference in chemical catalysis by low-affinity compared to high-affinity metal sites, some of these enzymes are also included in this article, especially if they are or have been discussed as zinc enzymes, or are active with zinc. 552 refs.

1,223 citations


Journal ArticleDOI
TL;DR: An automated refinement procedure (ARP) for protein models is proposed, and its convergence properties discussed, which are comparable to the iterative least-squares minimization/difference Fourier synthesis approach for small molecules.
Abstract: An automated refinement procedure (ARP) for protein models is proposed, and its convergence properties discussed. It is comparable to the iterative least-squares minimization/difference Fourier synthesis approach for small molecules. ARP has been successfully applied to three proteins, and for two of them resulted in models very similar to those obtained by conventional least-squares refinement and rebuilding with FRODO. In real time ARP is about ten times faster than conventional refinement. In its present form ARP requires high (2.0 A or better) resolution data, which should be of high quality and a starting protein model having about 75% of the atoms in roughly the correct position. For the third protein at 2.4 A resolution, ARP was significantly less powerful but nevertheless gave definite improvement, in the density map at least.

916 citations