The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.

The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.

Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL's chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

Designing a bed-side system for predicting length of stay in a neonatal intensive care unit.

Increased length of stay (LOS) in intensive care units is directly associated with the financial burden, anxiety, and increased mortality risks. In the current study, we have incorporated the association of day-to-day nutrition and medication data of the patient during its stay in hospital with its predicted LOS. To demonstrate the same, we developed a model to predict the LOS using risk factors (a) perinatal and antenatal details, (b) deviation of nutrition and medication dosage from guidelines, and (c) clinical diagnoses encountered during NICU stay. Data of 836 patient records (12 months) from two NICU sites were used and validated on 211 patient records (4 months). A bedside user interface integrated with EMR has been designed to display the model performance results on the validation dataset. The study shows that each gestation age group of patients has unique and independent risk factors associated with the LOS. The gestation is a significant risk factor for neonates < 34 weeks, nutrition deviation for < 32 weeks, and clinical diagnosis (sepsis) for ≥ 32 weeks. Patients on medications had considerable extra LOS for ≥ 32 weeks' gestation. The presented LOS model is tailored for each patient, and deviations from the recommended nutrition and medication guidelines were significantly associated with the predicted LOS.

Elucidating the Structural Features of ABCA1 in its Heterogeneous Membrane Environment.

ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The other theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular "reservoir". This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within the extracellular domains (ECDs) but not in the TMDs, giving it an outward-facing conformation. This structure was hypothesized to substantiate the lateral access theory. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a large heterogeneous lipid environment and found that the protein undergoes several large conformational changes in its extremities. We observed that the cavity enclosed within ATP unbound form of ABCA1 is narrow at the distal ends of TMD as well as the ECD region substantiating the "lateral access" theory. We have also characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.