Crystal structure of N-{N-[N-acetyl-(S)-leucyl]-(S)-leucyl}norleucinal (ALLN), an inhibitor of proteasome

The crystal structure of ALLN, the tripeptidic inhibitor of proteasomes, is solved from synchrotron diffraction data. An infinite β-sheet extended through the crystal is formed by symmetry-related oligopeptide molecules in extended conformation.


Chemical context
Proteasomes are high-molecular-mass multicatalytic enzyme complexes localized in the nucleus and cytosol of all eukaryotic cells. As a part of the ubiquitin-proteasome pathway, the complex executes a remarkable set of functions, ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling molecules (Adams, 2003;Groll & Potts, 2011). The ubiquitinproteasome pathway has been implicated in several forms of malignancy, in the pathogenesis of some autoimmune disorders, the aging process related cardiac dysfunction, diabetic complications, and neurodegenerative diseases (e.g. Alzheimer's, Parkinson's, Huntington's) (Dahlmann, 2007;Paul, 2008;Jankowska et al., 2013). Therefore, study of proteasome functions and the design and development of proteasome inhibitors is being pursued in many laboratories (Bennett & Kirk, 2008). A great amount of effort has been expended to explore proteasome inhibition as a novel targeted approach in cancer therapy. The first success came with FDA approval of Bortezomid for the treatment of multiple myeloma (Kane et al., 2006;Goldberg, 2012). Since then, numerous compounds have been reported to inhibit the components of the ubiquitin-proteasome system, and several new drug candidates undergoing clinical trials have emerged (Genin et al., 2010;Tsukamoto & Yokosawa, 2010;Frankland-Searby & Bhaumik, 2012;Jankowska et al., 2013). Peptide aldehydes were the first inhibitors designed to target the proteasome, and are still the most commonly used and best characterized group of such inhibitors (Kisselev et al., 2012). A notable one among them, Ac-Leu-Leu-Nle-H (ALLN, MG101), is also a potent inhibitor of nonproteasomal cysteine protease calpain I (Pietsch et al., 2010). ALLN, a cell-permeable tripeptide aldehyde reversible inhibitor of chymotripsin-like proteolytic activity of the proteasomes, was the first to be crystallized in a complex with an eukaryotic proteasome (Groll et al., 1997). Crystallographic analysis of the complex at 2.4 Å resolution revealed a structural organization of the proteasome and how the inhibitor binds to its active site. ALLN, as well as other peptide aldehydes, do it via reversible hemiacetal formation with the involvement of N-terminal threonine hydroxy group in the proteasome -subunits (Borissenko & Groll, 2007). The aldehyde structure derived from the crystal complex coordinates was used in molecular modeling of inhibitor-proteasome interactions (Zhang et al., 2009). High resolution structural data from this study may provide better accuracy in future modeling of the inhibitor interactions with proteasome and other potential intracellular targets.

Structural commentary
We report here the crystal structure of ALLN refined against 0.65 Å resolution diffraction data measured with synchrotron radiation. The molecule adopts an extended conformation of the backbone chain ( Fig. 1) with the ', -torsion angles residing in the region of the Ramachandran plot (Ramakrishnan & Ramachandran, 1965). All three consecutive peptide residues are in trans conformation and their ! angles are À179.42 (9), 173.77 (8), and 177.72 (10) . The side chains of the two leucine and one norleucine residues have unstrained conformations, and do not deviate by more than 7 from either trans or gauche rotamers along the consecutive C-C bonds.

Supramolecular features
All of the peptide ALLN N and O atoms are engaged in intermolecular hydrogen bonds (Table 1) between molecules related by the crystallographic 2 1 axis, forming an infinite antiparallel -sheet throughout the crystal (Fig. 2). The interactions between the sheets are mainly by the hydrophobic contacts of the aliphatic amino acid side chains. The arrangement of ALLN molecules in the ac plane, interacting through their aliphatic side chains, is illustrated in Fig. 3.

Synthesis and crystallization
The title aldehyde was prepared according to the general synthetic procedure reported by Schaschke et al. (1996), and a 45% overall yield was obtained. The product was crystallized from acetonitrile.

Figure 1
The molecule of ALLN, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level.

Figure 2
Backbones of three neighboring molecules of ALLN, forming a fragment of an antiparallel -sheet extending through the crystal. The amino acid side chains are not shown for clarity. Table 1 Hydrogen-bond geometry (Å , ).

Refinement details
Crystal data, data collection and structure refinement details are summarized in Table 2. A needle-like crystal elongated in the a direction was selected, picked up in the rayon loop and then quickly cryo-cooled in a stream of cold nitrogen gas at the single-axis goniostat of the SER-CAT synchrotron station ID19 at the Advanced Photon Source, Argonne National Laboratory, USA. Diffraction images were collected with the use of MAR300 CCD detector in two passes differing in the effective exposure and resolution limits in order to adequately measure the weakest high-resolution reflections, as well as the strongest low-angle reflections without overloading detector pixels. All 38117 measured intensities from both passes were integrated, scaled and merged by HKL-2000 (Otwinowski & Minor, 1997) into the set of 4561 unique reflections with the overall R merge factor of 0.049. The data set is rather strong, with the I/(I) ratio equal to 25 at the highest resolution of 0.65 Å . H atoms were located in a difference synthesis and refined as riding on their parent atoms in geometrically idealized positions. Because of the short wavelength of synchrotron radiation, all Friedel mates were averaged during data processing. The chirality of the molecule was deduced from the known chiral centres in the substrates used in chemical synthesis. Arrangement of ALLN molecules in the ac plane of the crystal, interacting through their aliphatic side chains.   program(s) used to refine structure: SHELXL (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windwows (Farrugia, 2012) and pyMOL (DeLano, 2002); software used to prepare material for publication: SHELXL97 (Sheldrick, 2008).

N-{N-[N-Acetyl-(S)-leucyl]-(S)-leucyl}norleucinal
Crystal data where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.44 e Å −3 Δρ min = −0.29 e Å −3 Special details Experimental. diffraction data were measured at the station 22ID of the APS synchrotron by rotation method a in three sweeps of different exposure and all data were scaled and merged into one data set Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against all reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on all data will be even larger.