**4. Conclusions**

nicotine acetylcholine receptor (hnAChR) [108] and *E. coli* mechanosensitive channel (MscL) [109]. hnAChRs are located mainly in the central nervous system and mediate fast neurotransmission. They possess a homopentameric quarternary structure where each monomer consists of an extracellular domain involving a conserved Cys loop and a ligand-binding channel, four transmembrane domains, and an intracellular segment. MscL is an integral membrane protein that gates in response to membrane tension in order to diminish the turgor pressure when bacteria are moved from a high to a low osmolarity environment. Like nAChR, MscL is a homopentamer where each subunit is composed of a cytoplasmic and a

*Homology Molecular Modeling - Perspectives and Applications*

Similar approaches were use in both studies. First a homology model of the target protein was constructed based on the known structures of similar proteins, followed by the application of NMA. The aim of both studies was to better understand channel gating and the particular structural changes associated with it [108, 109]. The

Enzymes have also been studied using a combination of HM and NMA. One example is the *Arabidopsis thaliana* Dicer-like 4 protein (AtDCL4) [110]. Dicer-like 4 is a large multidomain protein belonging to the Ribonuclease III family and is involved in the regulation of gene expression and antiviral defense through RNAinterference pathways. In particular, AtDCL4 produces short-stranded RNAs (tasiRNA) which are incorporated into the RNA-induced silencing complex to direct the silencing of cognate RNA [111, 112]. The main aim of the study [110] was to better understand the mechanism of AtDCL4-mediated dsRNA recognition and binding by which small RNAs of a specific size are produced. First, the authors built the core of the AtDCL4 protein, which consists of a Platform, a PAZ domain, a Connector helix and RNAseA/B domains. A model of an AtDCL4–dsRNA complex was then constructed, which suggested that the spatial orientation of the AtDCL4 domains with respect to one another are responsible for the length of the bound dsRNA. Two regions, one on the surface of the Platform domain and second in the PAZ loop, were also identified, which are likely to be responsible for RNA binding.

One of the longest-studied cellular processes is cell division [113] and cellular transport [114]. Regarding cell division, a combination of HM and NMA was used to study a yeast cohesin, an essential ring-shaped chromosome maintenance protein that mediates sister chromatid cohesion, homologous recombination, and DNA looping. This protein is a member of the structural maintenance of chromosomes (Smc) family, which exists in all eukaryotes [115]. In yeast, cohesin mainly consists of two Smc proteins, Smc1 and Smc3, both of which adopt long, anti-parallel coiled-coil regions that are separated by two globular regions: an ATP-binding head domain and a hinge region. The aim of the HM and NMA study [113] was to reveal the missing molecular details of how the two halves of the hinge region open to create an entry gate for DNA. In agreement with experimental data, the constructed yeast cohesin HM model showed that the bending motion of the cohesin ring is able to adopt a head-to-tail conformation. At the interface of the cohesin heterodimer, lowfrequency conformational changes were observed to deform the highly conserved glycine residues present there. Normal mode analysis further revealed that the docking of large globular structures, such as the nucleosome and accessory proteins, to cohesin notably affected the mobility of the coiled-coil regions. Moreover, fully

hnAChR study also involved predicting the ligand binding site [108].

transmembrane domain.

*3.2.3 Cell division and transport proteins*

*3.2.2 Enzymes*

**22**

Normal mode analysis is a very useful technique for determining which conformational states are accessible to a given macromolecule. It can provide much of the same information given by more computationally expensive methods, such as molecular dynamics simulation, at only a fraction of the cost. It can be used by itself or in tandem with HM to characterize the general flexibility and domain movements of a molecule, to produce possible alternative conformations and confirm observed ones, and to describe the conformational changes that occur or might occur during substrate binding, product release, or catalytic activation. We have illustrated its utility using several examples of its application to a number of large biologically important proteins and protein complexes from bacteria, eukaryotes, and viruses. The biological relevance of the *in silico* models constructed by HM and NMA can be verified and expanded by different experimental approaches involving molecular and structural biology and biochemistry.
