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.

The authors have nothing to disclose.

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 a critical step as it conditions the structural studies and further biochemical characterization (e.g., studying the dodecamer reassociation in various doses of Co2+). The molecular replacement, a proven method for phase determination, was used to solve the structures of TmPep1050 oligomers and its variants. The proposed protocol may be adapted to study other metallo-enzymes whose oligomerization states depend on the availability of their metal cofactors.
To illustrate the protocol, a case of study was presented, TmPep1050H60A H307A whose metal binding sites were impaired by mutating His-60 and His-307 to alanine. These residues bind Co2+ at the M2 and M1 sites, respectively. Interfering in metal binding could have perturbed the oligomerization state and led to a complete dissociation into monomers. Evidences of such a phenomenon have been reported for PhTET2 and PfTET3, two M42 aminopeptidases from P. horikoshii and P. furiosus, respectively24,29. TmPep1050H60A H307A did not behave as expected as this variant formed dimers only. Its structure showed the same modifications as the wild-type dimer but with two small exceptions. Indeed, the side chains of Asp-168 and Asp-62 appeared to be locked in an unconventional orientation preventing the stabilization of the active site. Their orientation seemed to be imposed by His-60 and His-307 as such modifications were not observed in the single point mutation variants.

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 apparent molecular weight, 52 kDa, has been reported for TmPep1050 dimer11. Hence, the oligomeric state of TmPep1050H60A H307A could be inferred as dimeric. Regarding its specific activity, TmPep1050H60A H307A was completely inactive on L-Leu-pNA as substrate, even in the presence of cobalt ions. This result strongly suggests that the variants cannot bind any metal ions.
The crystallization condition of TmPep1050H60A H307A was optimized by varying pH vs. PEG concentration (Figure 4) around the condition of the dimer (i.e., 0.1 M sodium citrate pH 6.0 10% PEG3350). The best crystals of TmPep1050H60A H307A were obtained in 0.1 M sodium citrate pH 5.2 20% PEG3350 with one cycle of microseeding for improving monocrystallinity. A complete data set was collected at Proxima 2 beamline (SOLEIL synchrotron) at a resolution 2.36 &#197; (Table 1). Data indexation showed that the space group of the TmPep1050H60A H307A crystal is C2221 but XDS proposed another solution, the mP space group (see Figure 5). According to Pointless, the likelihood of C2221 and P21 space groups were 0.711 and 0.149, respectively. According to the data quality analysis, two monomers are found in the asymmetric unit. The analysis by Xtriage revealed that the data set is probably twinned but twinning in C2221 space group is unlikely52. Twinning results from crystal growth anomaly where several definite domains have some of their lattice directions parallel to each other53. Twinning may also result from a higher crystal symmetry, indicating an erroneous data indexation. Hence, a pseudo-merohedral twin may exist so that a P21 crystal lattice looks like a C2221. The data set was subsequently indexed in space group P21 and tested in molecular replacement. Xtriage analysis of the data set indexed in P21 revealed a pseudo-merohedral twin following a twin law h, -k, -h-l.
Using the coordinates of a monomer from dodecameric TmPep1050 (PDB code 4P6Y), a molecular replacement solution was found for the data set indexed in P21 only, with a TFZ score of 28.9. Therefore, the diffraction data were treated as a twinned data set for model building. To minimize the bias of molecular replacement, a first model was built by using phenix.autobuild54,55. The structure of TmPep1050H60A H307A was completed after several cycles of automated and manual refinement in Phenix and Coot (Table 1 and Figure 6A). The structure confirmed the oligomeric state with an interface surface of 1,710 &#197;2 between both monomers and a &#916;iG of -16.2 kcal mol-1 as calculated by PDBe Pisa56. In comparison, the interface surface and &#916;iG of dimeric TmPep10502-mer is 1,673 &#197;2 and -16.7 kcal mol-1, respectively.
The structure of TmPep1050H60A H307A is highly similar to the wild-type dimer structure with RMS of 0.774 &#197; upon alignment. Importantly, the same structural modifications are observed in both structure: high flexibility of the &#945;8 and &#945;10 helices, disordered active site Gln-196&#8211;Val-202, and the displacement of Lys-229&#8211;Ala-235 and Lys-247&#8211;Ser 254. These modifications were correlated previously with the hindrance of dodecamer formation in absence of its metal cofactor11. The two mutations of His-60 and His-307, however, had a slight effect on the side chains of Asp-168 and Asp-62. They appeared to be locked in a conformation different from the wild-type dimer (Figure 6B). The Asp-168 carboxylate is rotated by 40&#176; due to the absence of His-60 and His-307. Hence both histidine residues are important for positioning the Asp-168 carboxylate correctly for bridging the two metal ions. The Asp-62 side chain is oriented towards Glu-18 carboxylate, outside the catalytic site. Asp-62 may have an important role in catalysis as it is assumed to modulate the pKa of His-60 and, thus, influence metal ion binding in the M2 site. In addition, it could be implicated structurally in the stabilization of the catalytic site upon metal ion binding, favoring the formation of the dodecamer.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61288/61288fig01.jpg" /> 
Figure 1: Schematic representation of TM_1050 ORF cloning into pBAD vector by homologous recombination. 
The ORF is flanked by two 30 bp sequences homologous to the promoter BAD end and the sequence upstream PmeI restriction site. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61288/61288fig02.jpg" /> 
Figure 2: Chromatograms of TmPep1050 purification. 
(A) Anion exchange chromatography. (B) Hydrophobic interaction chromatography. The absorbance (Abs), expressed in milliunits of absorbance (mUA), is shown in plain line. The conductivity, expressed in mS cm-1, is shown in dashed line. The grey box indicates where TmPep1050 eluates on the chromatograms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61288/61288fig03.jpg" /> 
Figure 3: Size exclusion chromatography of (A) TmPep1050 dodecamer, (B) TmPep1050H60A H307A, and (C) TmPep1050H60A. 
Samples were analyzed using SEC resin packed in a 120 mL column. Absorbance (Abs) is expressed in milliunits of absorbance (mUA). (D) Calibration of the SEC column using thyroglobulin (T), ferritin (F), aldolase (Ald), conalbumin (C), and albumin (Alb) as standards. The correlation between the logarithm of the relative mass and the elution volume is linear, with a R2 of 0.91. The 95% confidence intervals are represented as dots. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61288/61288fig04.jpg" /> 
Figure 4: Optimization of TmPep1050H60A H307A crystallization. 
(A) The first optimization strategy consists of varying pH (between 4.5 and 6.0) vs. PEG3350 concentration (between 5% and 25%). The crystallization plate is schematized, and the wells are color coded: red for precipitate, yellow for polycrystals, and green for monocrystals. (B) The second optimization strategy includes the use of seeds diluted 25x with a narrower variation of pH vs. PEG3350. (C) Crystal shape and size before (upper image) and after (lower image) crystallization optimization and microseeding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61288/61288fig05.jpg" /> 
Figure 5: Excerpts from the log output CORRECT.LP of TmPep1050H60A H307A data indexation by XDS. 
Upper panel, the possible Bravais lattices, the most likely being mC, mP, and oC. Middle panel, overall statistics of data indexed in C2221 space group. Lower panel, overall statistics of data indexed in P21 space group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61288/61288fig06.jpg" /> 
Figure 6: Structure of TmPep1050H60A H307A. 
(A) Structural alignment of a TmPep1050H60A H307A subunit (red, PDB code 5NE9) vs. a dodecamer subunit (white, PDB code 6NW5) and a dimer subunit (blue, PDB code 5NE6). Arrows indicate the structural dissimilarities between dodecamers and dimers. (B) Close-up of the TmPep1050H60A H307A active site (red) compared to the active site of TmPep1050 dimer (blue) and dodecamer (white). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61288/61288fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>TmPep1050H60A H307A</td>
		</tr>
		<tr>
			<td>Data collection</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Radiation source</td>
			<td>SOLEIL Proxima 2</td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>0.9801</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Dectris Eiger X 9M</td>
		</tr>
		<tr>
			<td>Oscillation range (&#176;)</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>Exposure time (s)</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>Space group</td>
			<td>P 1 21 1</td>
		</tr>
		<tr>
			<td>Unit cell parameters</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;, &#946;,&#160;&#947; (&#176;)</td>
			<td>90.00, 110.69, 90.00</td>
		</tr>
		<tr>
			<td>a, b, c (&#197;)</td>
			<td>43.24, 137.79, 61.11</td>
		</tr>
		<tr>
			<td>Resolution</td>
			<td>43.99 &#8211; 2.37 (2.52-2.37)</td>
		</tr>
		<tr>
			<td>Unique reflections</td>
			<td>26.902</td>
		</tr>
		<tr>
			<td>Rmerge (%)</td>
			<td>0.14</td>
		</tr>
		<tr>
			<td>Redundancy</td>
			<td>6,815</td>
		</tr>
		<tr>
			<td>&#60;I/&#963;&#62;</td>
			<td>8.64 (2.12)</td>
		</tr>
		<tr>
			<td>Completeness (%)</td>
			<td>99.6 (97.9)</td>
		</tr>
		<tr>
			<td>CC1/2 (%)</td>
			<td>99.2 (84.1)</td>
		</tr>
		<tr>
			<td>Refinement</td>
			<td></td>
		</tr>
		<tr>
			<td>Resolution</td>
			<td>43.99 &#8211; 2.37</td>
		</tr>
		<tr>
			<td>Reflections</td>
			<td>26.9</td>
		</tr>
		<tr>
			<td>Rfree set test count</td>
			<td>1345</td>
		</tr>
		<tr>
			<td>Rwork/Rfree</td>
			<td>0.206/0.234</td>
		</tr>
		<tr>
			<td>Protein molecules per ASU</td>
			<td>2</td>
		</tr>
		<tr>
			<td>VM (&#197;3/Da)</td>
			<td>2.37</td>
		</tr>
		<tr>
			<td>Solvent content (%)</td>
			<td>49.0</td>
		</tr>
		<tr>
			<td>Protein/solvent atoms</td>
			<td>4,559/96</td>
		</tr>
		<tr>
			<td>r.m.s.d. bond lengths (&#197;)</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>r.m.s.d. bond angles (&#176;)</td>
			<td>0.51</td>
		</tr>
		<tr>
			<td>Average B-factors (&#197;2)</td>
			<td>57.0</td>
		</tr>
		<tr>
			<td>Favored/disallowed Ramachandran &#966;/&#968; (%)</td>
			<td>95.02 / 0.17</td>
		</tr>
		<tr>
			<td>Twin law</td>
			<td>h, -k, -h-l</td>
		</tr>
		<tr>
			<td>PDB code</td>
			<td>5NE9</td>
		</tr>
	</tbody>
</table>
Table 1: Data collection and refinement statistics. Values in parentheses are for the highest-resolution shell.

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 ...

x-ray-crystallography-to-study-oligomeric-state-transition-thermotoga

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.9K Views.  Université Libre de Bruxelles. 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.

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

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

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

Summary

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Chapters in this video

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

Protein Crystallization for X-ray Crystallography