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

TLDR: 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.

Figures

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. This difference is about twice the ESD of the distance measurement at this resolution. Although a comparison of these two bond distances with each other may be unreliable because the difference between them is near the limits of their error estimates, a comparison can be made between the carbon–oxygen distances of Asp179 and those of Asp117 and Glu152. 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. The carbon–oxygen distance for the dangling oxygen compares well

Fig. 5. Proposed chemical reaction mechanisms for AAP

Table 1. Data processing and refinement statistics for the structures of native aminopeptidase from Aeromonas proteolytica at 0.95-Å resolution and at pH 4.5

Table 3. Zn–X distances (Å) of active-site metal ligands at 0.95-Å resolution

Fig. 4. A superposition of the active-site atoms of AAP with the active-site atoms of the AAP–l-leucinephosphonic acid (LPA) complex. The atoms in dark gray represent the active site for the native enzyme, while those in light gray represent the AAP–LPA complex. LPA has been stripped away for clarity

Fig. 1. 2F o−F c electron density map corresponding to the active-site amino acids contoured at 1.5σ. Electron density for Glu151 is not shown for clarity

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'e High-Resolution Structures of the Neutral and
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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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 Zn–O bond distances. A survey of the Cambridge Structural Database (CSD) [9] and ab initio calculations [10
]
have provided Zn–OH and Zn–OH
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

Frequently asked questions

Q1. What is the role of Zn2 in the bridging water molecule?

The role of Zn2 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. 

Q2. What are the contributions mentioned in the paper "The high-resolution structures of the neutral and the low ph crystals of aminopeptidase from <em>aeromonas proteolytica</em>" ?

Herein the authors report the crystal structures of AAP at 0. 95-Å resolution at neutral pH and at 1. 24-Å resolution at low pH. 

Q3. What was the use of CNS for the refinement of the structure?

The refinement program, CNS, was used for a rigid-body refinement using reflections from 30.0- to 4.0-Å resolution range and for several rounds of isotropic positional refinement using incrementally higher resolution data to 1.20 Å (1.24 Å for the low pH structure). 

Q4. Why were the positions of all the atoms not restrained during refinement?

Because the positions of all the atoms were not restrained during refinement, precise Zn–ligand and C–O distances could be obtained without bias. 

Q5. How does His256 shift away from the center of the active site?

To make way for the Zn1 movement, His256 shifts away from the center of the active site by 0.06 Å and rotates 2° around the C α –C β bond. 

Q6. What is the corresponding lack of electron density for hydrogen atoms?

The lack of electron density corresponding to hydrogen atoms is expected for the side chain oxygen atoms of aspartic acid and glutamic acid, and the nitrogen of histidine ligands, since their side chains are within the first and second coordination spheres of the two metal ions and the pH is above the individual pK as for the liganded amino acid side chains. 

Q7. What is the likely candidate to function as the nucleophile?

The reaction mechanism step at which deprotonation of the bridging solvent molecule occurs, the most likely candidate to function as the nucleophile, is unknown. 

Q8. What is the likely cause of the increase in the Zn1–O coordination distance?

The increase in the Zn1–O coordination distance may be the result of adding a proton to the bridging hydroxide ion, the change in the coordination number of Zn1 from 4 to 5, or both. 

Q9. What was the scale factor for the high-resolution data set?

The scale factor for the final image of the high-resolution data set was 0.72, while that for the low-resolution data set was 1.32. 

Q10. What is the average B iso value for all active-site amino acids?

The average B iso value for all of the active-site amino acids is 7.17 Å2 compared with 10.85 Å2 for all protein atoms, indicating the active-site amino acids are slightly more rigid with respect to the rest of the enzyme. 

Q11. How many peaks were associated with side chain carbons?

For the 30 identical peaks, 21 were associated with side chain carbons while only five were associated with C α and four with backbone nitrogen atoms. 

Q12. How many peaks were identified as potential hydrogen atoms?

At 0.95-Å resolution 237 peaks (approximately 100 more peaks than were found at 1.20-Å resolution) were identified as potential hydrogen atoms with 30 peaks previously identified at 1.20-Å resolution. 

Q13. What is the average Zn–O distance in the first coordination sphere?

The average Zn–N distance in the first coordination sphere of both metal ions is 2.03 Å and the average Zn–O distance is 2.07 Å, while in the second coordination sphere the average Zn–O distance is 2.39 Å (Table 3). 

Q14. What is the average Zn–O distance for the bridging oxygen species?

The Zn–O distances for the bridging oxygen species in this structure are 2.01 Å to Zn1 and 1.93 Å to Zn2, suggesting the bridging oxygen species is an OH−. 

Q15. What is the average B iso value for the ligands to Zn2?

The side chain residues liganded to Zn1 have an average B iso value of 8.47 Å2, which is significantly higher than the average B iso value for the ligands to Zn2 (7.01 Å2). 

Q16. What is the role of Zn(II) ions in the catalytic process?

Definitive assignment of the roles of each of the Zn(II) ions awaits a high-resolution structure of a productive enzyme–substrate complex, a difficult but worthwhile goal for this class of enzyme. 

Q17. What is the optimum distance of the metal ions to their ligands?

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.