Name: x-ray crystallography to study the oligomeric state transition of the thermotoga maritima m42 aminopeptidase tmpep1050
Uploaded: 2020-05-13T14:30:00
Description: Watch this Scientific Journal Video about X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050 at JoVE.com

We thank Martine Roovers for proofreading this paper and giving constructive comments. Access to Proxima 2 beamline (SOLEIL synchrotron) was within Block Allocation Groups 20151139.

1. Production and purification of recombinant TmPep1050
NOTE: Hereafter are described the cloning procedure and purification of wild-type TmPep1050 adapted from a previous study11. Alternatively, the cloning can be done using a synthetic gene. To generate TmPep1050 variants, site directed mutagenesis can be performed following, for instance, the single-primer reactions in parallel protocol (SPRINP) method30. The purification protocol can be used fo...

The protocol described herein allows understanding the dimer-dodecamer transition of TmPep1050 at the structural level. The methodology was experienced previously for determining the structure of both TmPep1050 oligomers11. The most challenging step was to find conditions promoting the dissociation of dodecamers into stable dimers. Such conditions had to be mild enough to permit the reassociation of dimers into dodecamers when the metal ion cofactor was added. The separation of oligomers was also ...

Oligomerization is a predominant process that dictates the biological functions of many proteins. In Escherichia coli, it is estimated that only 35% of proteins are monomeric1. Some proteins, called morpheeins, can even adopt several oligomeric states with subunits having distinct structure in each oligomeric state2. The transition between their oligomeric states is often a mean to regulate protein activity as each oligomeric state may have a different specific activity or function. Several examples of morpheeins have been well-documented in literature, notably the porphobilinogen synthase3, HPr kinase/phosphatase4, Lon protease5, lactate dehydrogenase6, glyceraldehyde-3-phosphate dehydrogenase7, pyruvate kinase8, citrate synthase9, and ribonuclease A10. Recently, we described the M42 aminopeptidase TmPep1050, another example of enzyme with morpheein-like behavior, whose activity depends on its oligomeric states11. The transition between its oligomeric states is mediated by its metallic cofactors which induce several structural modifications of the subunits.
The M42 aminopeptidase family belongs to the MH clan12,13, and is widely distributed among Bacteria and Archaea14. The M42 aminopeptidases are genuine dinuclear enzymes degrading peptides up to 35 amino acid residues in length15. They adopt a peculiar tetrahedron-shaped structure made of 12 subunits with their active sites&#160;oriented towards an inner cavity. Such an arrangement is often described as a nano-compartmentalization of the activity to avoid uncontrolled proteolysis. The physiological function of the M42 aminopeptidases may be associated with the proteasome, hydrolyzing peptides resulting&#160;from protein degradation16,17. Pyrococcus horikoshii possesses four M42 aminopeptidases, each presenting distinct but complementary specificities18,19,20,21. Singularly, heterocomplexes made of two different types of subunits have been described in P. horikoshii, suggesting the existence of peptidasome complexes22,23.
Several structures of M42 aminopeptidases have been described in the literature11,16,18,19,20,24,25,26. The subunit is composed of two distinct domains, a catalytic domain and a dimerization domain. The catalytic domain adopts a common &#945;/&#946; fold&#160;conserved in the whole MH clan, the archetypal catalytic domain being the aminopeptidase Ap1 of Vibrio proteolyticus27. The dimerization domain adopts a PDZ-like fold16 and may have, in addition to its role in the oligomerization, a role in controlling substrate access and binding in the inner cavity11. As the basic building block is a dimer, the dodecamer is often described as the association of six dimers, each dimer being positioned at each edge of the tetrahedron16. The oligomerization of the M42 aminopeptidases relies on the availability of its metal cofactors. Divalent metal ions, often Zn2+ and Co2+, are catalytically involved in the peptide binding and hydrolysis. They are found in two distinct binding sites, namely M1 and M2 sites. The two metal ions also drive and finely tune the oligomerization as demonstrated for PhTET2, PhTET3, PfTET3, and TmPep105011,24,28,29. When the metal cofactors are depleted, the dodecamer disassembles into dimers, like in PhTET2, PhTET3, and TmPep105011,16,28, or even monomers, like in PhTET2 and PfTET324,29.
Presented here is a protocol used for studying the structures of TmPep1050 oligomers. This protocol is a set of common methods including protein purification, proteolytic activity screening, crystallization, X-ray diffraction, and molecular replacement. Subtleties inherent to dealing with metalloenzymes, protein oligomerization, protein crystallization and molecular replacement are emphasized. A case of study is also presented to show whether TmPep1050 dodecamers may further dissociate into monomers or not. To address this question, a TmPep1050 variant, TmPep1050H60A H307A, has been studied whose metal binding sites are impaired by mutating His-60 (M2 site) and His-307 (M1 site) to Ala residues. This protocol may be accommodated to study other M42 aminopeptidases or any metalloenzymes with morpheein-like behavior.

<table>
	<tbody>
		<tr>
			<td>1,10-phenanthroline</td>
			<td>Sigma-Aldrich</td>
			<td>13, 137-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 ml Centrifugal Filters Ultracel 30K</td>
			<td>Merck Millipore</td>
			<td>UFC503096</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 15 Centrifugal Filters Ultracel 30K</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Merck Millipore</td>
			<td>70664-3</td>
			<td></td>
		</tr>
		<tr>
			<td>CCP4</td>
			<td>N/A</td>
			<td></td>
			<td>visit http://www.ccp4.ac.uk/</td>
		</tr>
		<tr>
			<td>cOmplete EDTA-free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coot</td>
			<td>N/A</td>
			<td></td>
			<td>visit https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td>
		</tr>
		<tr>
			<td>Crystal Screen I</td>
			<td>Hampton Research</td>
			<td>HR2-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Screen II</td>
			<td>Hampton Research</td>
			<td>HR2-112</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq Green PCR Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>K1082</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyXtal 15-well tool</td>
			<td>NeXtal</td>
			<td>132007</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli PPY strain</td>
			<td>N/A</td>
			<td></td>
			<td>see reference 31</td>
		</tr>
		<tr>
			<td>Escherichia coli XL1 blue strain</td>
			<td>Agilent</td>
			<td>200249</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Filtration Calibration Kit HMW</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-4038-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Filtration Calibration Kit LMW</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-4038-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Filtration Standard</td>
			<td>Biorad</td>
			<td>1511901</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Index</td>
			<td>Hampton Research</td>
			<td>HR2-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Litholoops</td>
			<td>Molecular Dimensions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine-p-nitroanilide</td>
			<td>Bachem AG</td>
			<td>40010720025</td>
			<td></td>
		</tr>
		<tr>
			<td>Natrix 1</td>
			<td>Hampton Research</td>
			<td>HR2-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Natrix 2</td>
			<td>Hampton Research</td>
			<td>HR2-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neggia plugin</td>
			<td>Dectris</td>
			<td>N/A</td>
			<td>visit https://www.dectris.com/</td>
		</tr>
		<tr>
			<td>NeXtal Tubes JCSG Core Suite I</td>
			<td>NeXtal</td>
			<td>130724</td>
			<td></td>
		</tr>
		<tr>
			<td>NeXtal Tubes JCSG Core Suite II</td>
			<td>NeXtal</td>
			<td>130725</td>
			<td></td>
		</tr>
		<tr>
			<td>NeXtal Tubes JCSG Core Suite III</td>
			<td>NeXtal</td>
			<td>130726</td>
			<td></td>
		</tr>
		<tr>
			<td>NeXtal Tubes JCSG Core Suite IV</td>
			<td>NeXtal</td>
			<td>130727</td>
			<td></td>
		</tr>
		<tr>
			<td>pBAD-TOPO</td>
			<td>ThermoFisher Scientific</td>
			<td>K430001</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenix</td>
			<td>N/A</td>
			<td></td>
			<td>visit https://www.phenix-online.org/</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>Salt RX 1</td>
			<td>Hampton Research</td>
			<td>HR2-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Salt RX 2</td>
			<td>Hampton Research</td>
			<td>HR2-109</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 3.5K MWCO</td>
			<td>ThermoFisher Scientific</td>
			<td>88242</td>
			<td></td>
		</tr>
		<tr>
			<td>Source 15Phe</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17014702</td>
			<td></td>
		</tr>
		<tr>
			<td>Source 15Q</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17094705</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 prep grade</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17104301</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermotoga maritima MSB8 strain</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 43589</td>
			<td></td>
		</tr>
		<tr>
			<td>TmCD00089984</td>
			<td>DNASU Plasmid Repository</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>XDS</td>
			<td>N/A</td>
			<td></td>
			<td>visit http://xds.mpimf-heidelberg.mpg.de/</td>
		</tr>
		<tr>
			<td>xdsme</td>
			<td>N/A</td>
			<td></td>
			<td>visit https://github.com/legrandp/xdsme</td>
		</tr>
	</tbody>
</table>

x-ray crystallography to study the oligomeric state transition of the thermotoga maritima m42 aminopeptidase tmpep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

To study a possible dodecamer dissociation into monomers in TmPep1050, the His-60 and His-307 codons were replaced by alanine codon using a synthetic gene. This gene was then cloned in pBAD vector for expression and purification of the corresponding TmPep1050 variant subsequently named TmPep1050H60A H307A. Size exclusion chromatography (Figure 3B) showed that the purified protein had an apparent molecular weight of 56 kDa (molecular weight of the monomer being 36.0 kDa). A similar...

Watch this Scientific Journal Video about X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050 at JoVE.com

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

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