## 2 Structure of the USP Family  
Most of the information on the biochemical and physiological attributes of Ups stem from being studied in _E. coli_[24]. The USP domain is a highly conserved 140-160 amino acid-containing domain that may exist in one of three forms: as a single 14-15 kDa domain that makes up the whole protein, as a tandem domain of approximately 30 kDa within larger USP proteins on a single polypeptide chain, or as two Usp domains fused together with catalytic domains that include Cl\({}^{-}\) voltage channels, Na\({}^{+}/\)H\({}^{+}\) antiporter domains, protein kinase domains or amino acid permeases (Figure 1) [15; 25; 26]. A large contributing factor to the ability of USPs to resist multiple stresses is the structural diversity associated with the functional motifs attached to the USP domain caused by the process of evolution, which may have resulted in the attachment of these motifs that hold various biochemical and molecular consequences [3]. _E. coli_ USPs also develop homodimer and heterodimer  
Figure 1: Classification, domain composition and functions of known USPs. The figure depicts the multifaceted but overlapping functions of USPs and the form in which they exist in nature. formations with each other, therefore contributing to the different functional responses they display to a variety of stresses. USPs A, C and D form both homo- and hetero-dimers, while USPs F and G have the ability to form three types of dimers (Figure 1) [21]. USPs are categorized according to their ability to bind ATP; UspA and UspA-like proteins do not bind ATP, while USPs of the F and G-type bind ATP [27; 28]. In most cases, USPs that comprise attached domains belong to the FG type. These include USPs F (ynaF) and G (ybdQ) (Class II) as their names suggest, while USPs A, C (vecG) and D (yiiT) (Class I) form part of the UspA subfamily [29]. This subfamily, also known as the USP family (Pfam 00582), falls under the PP-loop clan (CL0039) as categorized by the Pfam database, which consists of 12 members; ATP_bind_3 and ATP_bind_4, Electron transfer flavoprotein, Arginosuccinate synthase, Thiamine biosynthesis protein (Thil), NAD synthase, Asparagine synthase, ExsB, tRNA methyl transferase, Phosphoadenosine phosphosulfate reductase family, DNA photolyase, and lastly the USPs [14]. The PP-motif exhibits different characteristics among clan members, resulting in a wide range of enzymatic and functional activities. It has been proposed that this domain in turn falls under the \(\alpha/\beta\) domain referred to as the HIGH-signature proteins, Usp-like domains and PP-ATPase (HUP) domains, which is an ancient and diverse group of proteins that contains photolyases, Usps and electron transfer flavoproteins, believed to have evolved from UspA-like proteins by losing their ATPase and nucleotide-binding functions. This has suggested that UspA-like proteins are actually ancestors of UspFG-like ATPase-binding proteins [7; 30]. The domains present in this group are mostly small domains assembled as Rossmann fold-like structures consisting of three layers containing five parallel \(\beta\)-sheets with two flanking \(\alpha\)-helices on each side [31]. The Structural Classification of Proteins database additionally categorizes USPs under the adenine nucleotide alpha hydrolases-like superfamily, which is in turn described by the superfamily database as possessing general functions, especially in nucleotide transport and metabolism [14]. The class is categorized by having an adenine nucleotide alpha hydrolase-like fold made up of assembled Rossmann fold-like structures consisting of five parallel \(\beta\)-sheets with two flanking \(\alpha\)-helices on each side and a three-layer \(\alpha/\beta/\alpha\) core in its interior [14; 31]. This fold is in turn placed within the \(\alpha/\beta\) class that is mainly made up of \(\beta/\alpha/\beta\) units. Classes III and IV are composed of two UspE (ydaA) units, namely E1 and E2 [6; 32; 33].