Hasan DeMirci

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus ( Gk _Lip ). The 2.47-Å orthorhombic model contains a Ca²⁺ ion at the conserved metal-binding site, whereas the 2.30-Å anisotropic triclinic model contains four non-equivalent protein chains in which no Ca²⁺ ion was modeled. Gk _Lip retains the canonical α/β-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn²⁺-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca²⁺ coordination environment. Pro367 adopts a Cγ-endo pucker in the Ca²⁺-containing structure; three Ca²⁺-free chains also adopt the endo pucker, whereas one adopts a Cγ-exo pucker. The presence of both Pro367 pucker states in the Ca²⁺-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus (GkLip). The 2.47-Å orthorhombic model contains a Ca2+ ion at the conserved metal-binding site, whereas the 2.30-Å anisotropic triclinic model contains four non-equivalent protein chains in which no Ca2+ ion was modeled. GkLip retains the canonical α/β-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn2+-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca2+ coordination environment. Pro367 adopts a Cγ-endo pucker in the Ca2+-containing structure; three Ca2+-free chains also adopt the endo pucker, whereas one adopts a Cγ-exo pucker. The presence of both Pro367 pucker states in the Ca2+-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Over the past two decades, drug discovery has undergone a remarkable transformation, evolving from a predominantly empirical endeavor into an increasingly rational and multidisciplinary scientific enterprise

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thermoacidophilic Crenarchaeota, like Metallosphaera sedula and Sulfolobus tokodaii, employs AMP-forming Acyl-CoA synthetases of the ANL superfamily to activate its key carboxylate intermediates. This is an energetically costly mechanism that consumes two ATP equivalents , a critical distinction from the energy-saving ADP-forming (ACD) enzymes found in Thaumarchaeota. To define the structural principles of this "AMP-forming solution," we targeted three specific crenarchaeal enzymes: M. sedula 4-hydroxybutyryl-CoA synthetase (Msed_0406), M. sedula 3-hydroxypropionyl-CoA synthetase (Msed_1456), and S. tokodaii 3-hydroxypropionyl-CoA synthetase (Stok_3HPCS). This work describes the protocols we developed for their heterologous expression and purification. While Msed_0406 and the thermostable Stok_3HPCS were successfully purified, Msed_1456 proved to be highly unstable and prone to degradation. Using a combined in vitro and in silico approach, we demonstrate this instability is an intrinsic feature linked to its catalytic mechanism. AlphaFold 3 models, validated by our purification data, reveal that while both Msed_1456 and Stok_3HPCS are homotetramers, the thermostable Stok_3HPCS buries a massive interface (14,360 Ų) while the unstable Msed_1456 buries a minimal interface (7,097 Ų). This flexibility is essential for the ANL mechanism, which we identify as a "phosphate-gated latch". We show the gamma-phosphate of ATP tethers the N- and C-terminal domains; its hydrolysis upon adenylation "un-latches" the enzyme, permitting a massive ~125° domain rotation to the thioesterification state. This work defines the AMP-forming solution to carboxylate activation, providing a critical counterpart to the rigid, energy-efficient ACD enzymes and offering foundational knowledge for engineering novel carbon-fixation pathways..

Elucidating the structure and dynamics of insulin and its analogs has been of broad interest, while presenting challenges due to the unique structural dynamics of insulin (composed itself of two multiply cross-linked peptides A and B) and its ability to assemble in a variety of oligomeric structures under physiological conditions. Here, we present two distinct X-ray crystallographic structures of the long-acting human insulin analog detemir (INSD) resolved in hexameric and dodecameric (or dihexameric) states at 2.85 Å and 2.70 Å resolution, respectively, using diffraction data collected under ambient temperature conditions. Characterization of the collective dynamics of these oligomers using the Gaussian Network Model (GNM) reveals several key features: (i) Oligomerization imparts high cooperativity in structural dynamics evidenced by dissection of the crosscorrelations at various hierarchical levels; (ii) detemir monomers conformational flexibility is highly suppressed within oligomeric constructs, the effect being particularly strong in the dihexamer due to the asymmetric packing of the hexamers and the presence of myristoyl groups at B peptides termini whose interactions imparts further heterogeneities; and (iii) a number of key residues retain, however, their intrinsic dynamics, to be deployed upon release from the oligomers. We distinguish in particular residues serving as hinge sites that mediates the conformational dynamics of the asymmetric units (dimers) and monomers (I2A-V3A and Y19A-C20A, and L11B-L15B and Y26B of the respective peptides A and B), or as anchors supporting structural stability (disulfide-bridge forming cysteines, plus selected residues such as L16A, G8B and R22B-F24B. Overall, this study provides a structural-dynamic framework for gaining new insights into the dynamics of long-acting analog INSD and helps identify actionable sites for modulating insulin (analogs) dynamics toward designing more effective therapeutics.

Abelson-1 (ABL-1) is a nonreceptor tyrosine kinase that plays essential roles in various cellular processes, including proliferation, survival, differentiation and its kinase activity is tightly regulated. The dysregulated ABL-1 kinase activity is linked to disease pathogenesis like Chronic Myeloid Leukemia (CML), where the BCR::ABL-1 fusion oncoprotein drives oncogenic signaling. Due to its central role in CML pathogenesis, understanding the structure of ABL-1 is crucial for the effective management of the disease and drug development studies. This study focuses on optimizing the expression, purification and crystallization of the recombinant human ABL-1 kinase domain for its structural analysis via X-ray crystallography and structure-based drug screening applications. The human ABL-1 kinase domain, fused with a SUMO-tag, was expressed in Escherichia coli Rosetta2 BL21 using the pET28(a)+ expression vector. The ABL-1 aggregates seen under native culture conditions were successfully solubilized by the mild ionic detergent sarkosyl. After obtaining soluble expression of the protein, Ni-NTA affinity chromatography was performed and high yield of purified ABL-1 was obtained. The 6X-His-SUMO-tag of purified ABL1 was cleaved by ULP1 protease. The recombinant ABL-1 was subsequently used in crystallization trials to enlighten structural features of ABL-1 that could guide the development of novel therapeutics and drug screening platforms targeting ABL-1 in CML.

The epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) is a key regulator of intracellular signaling events that control cell proliferation. Aberrant EGFR activation is closely associated with the development of non-small cell lung cancer (NSCLC). In this study, recombinant EGFR-TKD was expressed in Escherichia coli Rosetta™ 2 (DE3) cells using a pET28a(+) expression plasmid carrying an N-terminal 6×His-SUMO- tag. During expression, a large proportion of the protein accumulated in inclusion bodies; therefore, we employed a solubilization approach. Treatment with 1.5% sarcosyl reproducibly yielded a soluble protein fraction compatible with subsequent purification steps. Optimizing induction parameters, including temperature, IPTG concentration, and expression duration, improved both the yield and solubility of the protein. Following size-exclusion chromatography, the monomeric protein fraction was isolated and used for crystallization trials, which resulted in the formation of small but well-defined microcrystals under several conditions. Although these crystals did not yet provide diffraction suitable for structure determination, their reproducible appearance indicates that the obtained EGFR-TKD is structurally competent for crystallization trials. Overall, the workflow establishes a practical and reproducible bacterial expression and purification strategy that forms a basis for continued crystallization optimization and structure-based inhibitor development targeting EGFR-driven cancers.

Offshore wind power technology presents a viable solution to address significant challenges related to energy and climate change. Moreover, it has the potential to stimulate economic growth and create employment opportunities. Considering Türkiye's favourable geographical location and extensive coastline, the integration of offshore wind power stands as a pivotal factor in helping the country attain its energy targets from 2035 to 2053. There are currently no installed offshore wind turbines (OWTs) in Türkiye; however, the Turkish Ministry of Energy and Natural Resources has delineated three development zones in the Sea of Marmara for the installation of OWTs. With the commencement of developments in these specified areas, strategic plans aim to increase the installed capacity of OWTs. This study is an attempt to evaluate possible and plausible locations of wind turbine installation within Turkish maritime boundaries, which are estimated to have a high potential for offshore wind energy. Moreover, types of foundations supporting OWTs along with their associated design criteria are enlisted. Considering Türkiye's position in a seismically active region, an overview of seismic hazards is summarized, and the impact of relevant seismic hazards on foundations is discussed. It should be emphasized that, despite the drawbacks due to tourism and international seaborne traffic, Türkiye, having a population more than 85 million, has a great wind energy potential, which needs extensive and meticulous assessment for a sustainable future.