One Polyketide Synthase, Two Distinct Products: Trans-Acting Enzyme-Controlled Product Divergence in Calbistrin Biosynthesis.

Calbistrins are fungal polyketides consisting of the characteristic decalin and polyene moieties. Although the biosynthetic gene cluster of calbistrin A was recently identified, the pathway of calbistrin A biosynthesis has largely remained uninvestigated. Herein, we investigated the mechanism by which the backbone structures of calbistrins are formed, by heterologous and in vitro reconstitution of the biosynthesis and a structural biological study. Intriguingly, our analyses revealed that the decalin and polyene portions of calbistrins are synthesized by the single polyketide synthase (PKS) CalA, with the aid of the trans-acting enoylreductase CalK and the trans-acting C-methyltransferase CalH, respectively. We also determined that the esterification of the two polyketide parts is catalyzed by the acyltransferase CalD. Our study has uncovered a novel dual-functional PKS and thus broadened our understanding of how fungi synthesize diverse polyketide natural products.

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca2+-free protein. In contrast, the linker length has only a marginal effect on the Ca2+ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca2+ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca2+-bound state. For the extreme case of disconnected subdomains, Ca2+ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca2+ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.

Examination of morphological and synaptic features of calbindin-immunoreactive neurons in deep layers of the rat olfactory bulb with correlative laser and volume electron microscopy.

The olfactory bulb (OB) contains various interneuron types that play key roles in processing olfactory information via synaptic contacts. Many previous studies have reported synaptic connections of heterogeneous interneurons in superficial OB layers. In contrast, few studies have examined synaptic connections in deep layers because of the lack of a selective marker for intrinsic neurons located in the deeper layers, including the mitral cell layer, internal plexiform layer (IPL) and granule cell layer. However, neural circuits in the deep layers are likely to have a strong effect on the output of the OB because of the cellular composition of these regions. Here, we analyzed the calbindin-immunoreactive neurons in the IPL, one of the clearly neurochemically defined interneuron types in the deep layers, using multiple immunolabeling and confocal laser scanning microscopy combined with electron microscopic three-dimensional serial-section reconstruction, enabling correlated laser and volume electron microscopy (EM). Despite a resemblance to the morphological features of deep short axon cells, IPL calbindin-immunoreactive (IPL-CB-ir) neurons lacked axons. Furthermore, multiple immunolabeling for plural neurochemicals indicated that IPL-CB-ir neurons differed from any interneuron types reported previously. We identified symmetrical synapses formed by IPL-CB-ir neurons on granule cells (GCs) using correlated laser and volume EM. These synapses might inhibit GCs and thus disinhibit mitral and tufted cells. Our present findings indicate, for the first time, that IPL-CB-ir neurons are involved in regulating the activities of projection neurons, further suggesting their involvement in synaptic circuitry for output from the deeper layers of the OB, which has not previously been clarified.

Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D9k.

Calbindin D9k is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I. There is a significant discrepancy between simulated and experimental order parameters for site I residues in the case of no ion bound in site I. However, it was found in the simulations that a Na+ ion can bind in site I, and the resulting order parameters determined from the simulations are in excellent agreement with experiment. Comparisons are made to X-ray structures of other S100 family members in which Na+ ions were observed or suggested to be bound in site I. © 2019 Wiley Periodicals, Inc.

Large enhancement of response times of a protein conformational switch by computational design.

The design of protein conformational switches-or proteins that change conformations in response to a signal such as ligand binding-has great potential for developing novel biosensors, diagnostic tools, and therapeutic agents. Among the defining properties of such switches, the response time has been the most challenging to optimize. Here we apply a computational design strategy in synergistic combination with biophysical experiments to rationally improve the response time of an engineered protein-based Ca2+-sensor in which the switching process occurs via mutually exclusive folding of two alternate frames. Notably, our strategy identifies mutations that increase switching rates by as much as 32-fold, achieving response times on the order of fast physiological Ca2+ fluctuations. Our computational design strategy is general and may aid in optimizing the kinetics of other protein conformational switches.

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments ­ (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH ­ which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins.

Combining fragment homology modeling with molecular dynamics aims at prediction of Ca²âº binding sites in CaBPs.

The family of calcium-binding proteins (CaBPs) consists of dozens of members and contributes to all aspects of the cell's function, from homeostasis to learning and memory. However, the Ca²âº-binding mechanism is still unclear for most of CaBPs. To identify the Ca²âº-binding sites of CaBPs, this study presented a computational approach which combined the fragment homology modeling with molecular dynamics simulation. For validation, we performed a two-step strategy as follows: first, the approach is used to identify the Ca²âº-binding sites of CaBPs, which have the EF-hand Ca²âº-binding site and the detailed binding mechanism. To accomplish this, eighteen crystal structures of CaBPs with 49 Ca²âº-binding sites are selected to be analyzed including calmodulin. The computational method identified 43 from 49 Ca²âº-binding sites. Second, we performed the approach to large-conductance Ca²âº-activated K⁺ (BK) channels which don't have clear Ca²âº-binding mechanism. The simulated results are consistent with the experimental data. The computational approach may shed some light on the identification of Ca²âº-binding sites in CaBPs.

Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins.

Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone ((1)H, (15)N, (13)C(α) and (13)C') resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment--(4,3)D-hnCOCANH and exploits the linear combinations of backbone ((13)C(α) and (13)C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks--which is different in sum (CA + CO) and difference (CA - CO) frequency regions--greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide (15)N and backbone (13)C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) (15)N and (13)C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F2-F3 planes of the spectrum corresponding to sum (CA + CO) and difference (CA - CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.