Having been sitting on an X-ray crystal structure and half-finished manuscript for quite some time now, imagine my chagrin when during a casual perusal of the PDB, I found an extremely similar structure had just been deposited to the one I was aiming to publish.
The deposited structure (5X2L) is for wild-type human serine racemase (SR) and the associated paper focuses on in silico screening and medicinal chemistry, while my structure contains two cysteine-to-aspartate point mutations (C2D, C6D) with a (speculative) paper that explores the crystallography and biophysical side. As well as briefly summarising the findings of the study, I thought it would be fun to compare our two crystal structures in what I have dubbed…
Clash of the Crystals
But firstly, some background on the contestant/s: 5X2L (published) vs. CRK1 (mine, unpublished).
In the blue corner…(and the red corner)…weighing in at 37.4 kDa, we have a pyridoxial-5’-phosphate (PLP)-dependent enzyme that catalyses the racemisation of L-serine to D-serine, as well as the α,β-elimination of water from L-serine. This forebrain-localised enzyme produces about 90% of D-serine in the brain, and because D-serine is a co-agonist of N-methyl-D-aspartate glutamate receptors (NMDARs), SR inhibition has been touted as an up-and-coming approach to indirectly modulate NMDAR activity. This is a potential game-changer for treating disorders underpinned by NMDAR overactivation, such as neuropathic pain, neurodegenerative disorders, and epileptic states.
So let’s hear it for…serine racemase! [Thunderous applause]
Round 1: Paper summary
Anyone well versed in SR literature will know the paper in question, ‘Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase’ by Takahara et al. (2018)1 is an additional chapter to an ongoing story. Several groups have previously tried to identify new SR inhibitors that are potent, selective, and structurally distinct from the countless amino acid analogue inhibitors that are already well-described2, and for many this has proved to be a challenging endeavour.
The status quo shifted when a series of dipeptide-like inhibitors with a clear structural motif and slow-binding kinetics was identified by Dixon et al. (2006)3, which later provided the query molecule for an in silico screen performed by the same group behind 5X2L4. The resulting inhibitors contained an essential central amide structure with a phenoxy substituent, and substitution of parts of the structure for heavier halogen atoms such as bromine and iodine produced derivatives with improved inhibitory activity (comparable to classical SR inhibitors), binding affinity, and ligand efficiency. The Mori group took their explorations even further by testing the most potent derivative in vivo to demonstrate the SR inhibitor suppressed neuronal activity-dependent Arc expression to regulate NMDAR overactivation5.
The current paper expands on these studies by firstly, solving the crystal structure of wild-type SR for molecular docking and in silico screening; secondly, using these methods to identify new SR inhibitors related to their previously described peptide compounds; and thirdly, testing these inhibitors in an in vitro assay. The team synthesised 15 derivatives, of which one showed relatively high inhibitory activity, making a nice addition to their growing rolodex of peptide SR inhibitors.
Figure 1. Structure and binding pocket of the novel peptide SR inhibitor derivative identified by Takahara et al.
Round 2: Clash of the Crystals!
Both contestants were crystallised using the sitting-drop vapour diffusion method in very similar experimental conditions (Table 1). Both structures were determined to a highly respectable resolution of 1.8 Å, and organised into a large domain and small domain connected by a flexible loop region (Fig. 2). The PLP cofactor (Fig. 2; yellow sticks), on which SR is dependent for its catalytic activity, can be seen covalently linked to Lys56.
Table 1. Summary of key features of 5X2L and CRK1.
||10% PEG 8000, 5 mM MgCl2, 0.1 M Bis-Tris pH 6.0, 10% ethylene glycol, 20 °C
||15% PEG 3350, 250 mM MgCl2, 0.1 M Bis-Tris pH 6.5, 20 °C
|a, b, c (Å)
α, β, γ (°)
|80.1 112.6 88.0
90.0 90.0 90.0
|69.0 53.8 79.4
90.0 106.1 90.0
|No. residues resolved
SR belongs to the fold-type II family of PLP-dependent enzymes, meaning it contains a β-sheet core surrounded by α-helices, with the active site located in a cleft between the two domains. Accordingly, both domains of 5X2L and CRK1 contain a parallel-stranded β-sheet surrounded by nine α-helices in the large domain and three in the small domain. A magnesium ion (pink sphere) that helps to stabilise protein folding and increase maximal activity6 is octahedrally coordinated by three water molecules, the acid groups of Glu210 and Asp216, and the carbonyl oxygen of Ala214.
Figure 2. Overall X-ray crystal structure of the human SR holoenzyme CRK1. Residues are coloured from red to violet, N-terminus to C-terminus, and all helices are numbered 1–12 based on the order they occur in the polypeptide sequence. Each SR monomer comprises a large domain (helices 1–3 and 7–12) and small domain (helices 4–6) connected by a flexible loop region.
CRK1 boasts good ordering of residues, with only a few not well-defined: 1–3, 132–135, and 339–340. Aside from those at the C- and N-terminus, which are often poorly resolved during structure solution anyway, the only other undefined residues (132–135) were localised to the top of helix 5 in the highly-mobile small domain. 5X2L shows similarly undefined residues at the termini (1–2, 318–340) although in addition it is also missing residues of the flexible loop region (67–76) that connects the two domains.
Solvent-exposed loops are notorious for being tricky to model due to their high occupancy. The loop may be visible in CRK1 but not 5X2L because CRK1 has the help of its (symmetry) mates. By viewing the symmetry partners in the crystal lattice, the loop residues 69–73 are seen to be stabilised by a water-mediated interaction between Leu72 in one monomer and Lys221 in an adjacent monomer.
These favourable contacts may not occur for both structures because 5X2L crystallised in the orthorhombic space group P212121 while CRK1 crystallised in the monoclinic space group P21. Both possess 2-fold symmetry, but differences in molecular packing would have influenced whether the loop region would be suitably positioned to receive stabilising crystal lattice contacts.
Round 3: Best [Super]Pose!
A superposition of 5X2L and CRK1 (Fig. 3) revealed that, unsurprisingly, the two structures were well aligned with a Cα RMSD of 0.55 Å. Any remaining conformational differences are likely to result from the unresolved loop region, the missing helix and polypeptide strand that make up residues 318–340, and random structural variations. So hardly a ‘clash’ but at least it makes for a nice picture.
Figure 3. Superposition of the X-ray crystal structures of 5X2L (blue) and CRK1 (red).
By now there is no doubt you are wondering who the champion is of the undeniably riveting Clash of the Crystals.
The answer is both, and neither, because any discovery that contributes to scientific advancement is a champion in my book J
Yes, even when said discovery beats me to the punch.
Blog written by Chloe Koulouris
- Takahara S, Nakagawa K, Uchiyama T, Yoshida T, Matsumoto K, Kawasumi Y et al. Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase. Bioorg Med Chem Lett. 2017 Dec 13.
- Jirásková-Vaníčková J, Ettrich R, Vorlová B, Hoffman HE, Lepšík M, Jansa P et al. Inhibition of human serine racemase, an emerging target for medicinal chemistry. Curr Drug Targets. 2011 Jun; 12(7):1037-55.
- Dixon SM, Li P, Liu R, Wolosker H, Lam KS, Kurth MJ et al. Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries. J Med Chem. 2006 Apr; 49(8):2388-97.
- Mori H, Wada R, Li J, Ishimoto T, Mizuguchi M, Obita T et al. In silico and pharmacological screenings identify novel serine racemase inhibitors. Bioorg Med Chem Lett. 2014 Aug; 24(16):3732-5.
- Mori H, Wada R, Takahara S, Horino Y, Izumi H, Ishimoto T et al. A novel serine racemase inhibitor suppresses neuronal over-activation in vivo. Bioorg Med Chem. 2017 Jul 15; 25(14):3736-45.
- De Miranda J, Panizzutti R, Foltyn VN, Wolosker H. Cofactors of serine racemase that physiologically stimulate the synthesis of the n-methyl-d-aspartate (nmda) receptor coagonist d-serine. Proc Natl Acad Sci U S A. 2002 Oct; 99(22):14542-7.